2,4-disubstituted pyrimidines useful as kinase inhibitors

ABSTRACT

The present invention provides 2,4-disubstituted pyrimidine compounds useful as kinase inhibitors, pharmaceutically acceptable compositions thereof, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. Ser. No.12/492,180, filed Jun. 26, 2009, which claims priority to U.S.provisional application Ser. No. 61/076,450, filed Jun. 27, 2008, U.S.provisional application Ser. No. 61/148,388, filed Jan. 29, 2009, andU.S. provisional application Ser. No. 61/170,874, filed Apr. 20, 2009,the entirety of each of which is hereby incorporated by reference.

SEQUENCE LISTING

In accordance with 37 CFR 1.52(e)(5), a Sequence Listing in the form ofa text file (entitled “Sequence Listing.txt,” created on Dec. 29, 2009,and 36 kilobytes) is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors ofprotein kinases. The invention also provides pharmaceutically acceptablecompositions comprising compounds of the present invention and methodsof using said compositions in the treatment of various disorders.

BACKGROUND OF THE INVENTION

The search for new therapeutic agents has been greatly aided in recentyears by a better understanding of the structure of enzymes and otherbiomolecules associated with diseases. One important class of enzymesthat has been the subject of extensive study is protein kinases.

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within the cell. Protein kinases are thought tohave evolved from a common ancestral gene due to the conservation oftheir structure and catalytic function. Almost all kinases contain asimilar 250-300 amino acid catalytic domain. The kinases may becategorized into families by the substrates they phosphorylate (e.g.,protein-tyrosine, protein-serine/threonine, lipids, etc.).

In general, protein kinases mediate intracellular signaling by effectinga phosphoryl transfer from a nucleoside triphosphate to a proteinacceptor that is involved in a signaling pathway. These phosphorylationevents act as molecular on/off switches that can modulate or regulatethe target protein biological function. These phosphorylation events areultimately triggered in response to a variety of extracellular and otherstimuli. Examples of such stimuli include environmental and chemicalstress signals (e.g., osmotic shock, heat shock, ultraviolet radiation,bacterial endotoxin, and H₂O₂), cytokines (e.g., interleukin-1 (IL-1)and tumor necrosis factor α (TNF-α)), and growth factors (e.g.,granulocyte macrophage-colony-stimulating factor (GM-CSF), andfibroblast growth factor (FGF)). An extracellular stimulus may affectone or more cellular responses related to cell growth, migration,differentiation, secretion of hormones, activation of transcriptionfactors, muscle contraction, glucose metabolism, control of proteinsynthesis, and regulation of the cell cycle.

Many diseases are associated with abnormal cellular responses triggeredby protein kinase-mediated events as described above. These diseasesinclude, but are not limited to, autoimmune diseases, inflammatorydiseases, bone diseases, metabolic diseases, neurological andneurodegenerative diseases, cancer, cardiovascular diseases, allergiesand asthma, Alzheimer's disease, and hormone-related diseases.Accordingly, there remains a need to find protein kinase inhibitorsuseful as therapeutic agents.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asinhibitors of one or more protein kinases. Such compounds have generalformulae I-a and I-b:

or a pharmaceutically acceptable salt thereof, wherein Ring A, Ring B,m, p, R^(x), R^(y), R^(v), W¹, W² and R¹ are as defined herein.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful for treating a variety of diseases,disorders or conditions, associated with abnormal cellular responsestriggered by protein kinase-mediated events. Such diseases, disorders,or conditions include those described herein.

Compounds provided by this invention are also useful for the study ofkinases in biological and pathological phenomena; the study ofintracellular signal transduction pathways mediated by such kinases; andthe comparative evaluation of new kinase inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts dose-response inhibition of phospho-plc gamma2 (p-plcgamma 2) with compound I-2 in Ramos Cells; and the results of compoundI-2 in a “washout” experiment.

FIG. 2 depicts dose-response inhibition of p-plc gamma2 with compoundI-4 in Ramos Cells; and the results of compound I-4 in a “washout”experiment.

FIG. 3 depicts dose response inhibition of p-plc gamma2 with compoundI-7 in Ramos cells; and the results of compound I-7 in a “washout”experiment.

FIG. 4 depicts dose response inhibition of p-plc gamma2 with compoundI-35 in Ramos cells.

FIG. 5 depicts dose response inhibition of p-plc gamma2 with compoundI-38 in Ramos cells.

FIG. 6 depicts MS analysis confirming covalent modification of TECkinase at Cys449 by compound I-2.

FIG. 7 depicts MS analysis confirming covalent modification of TECkinase at Cys449 by compound I-4.

FIG. 8 depicts MS analysis confirming covalent modification of TECkinase at Cys449 by compound I-7.

FIG. 9 depicts the results of compound I-2 in a “washout” experiment ascompared to results of compound I-4 and compound I-7 in the same“washout” experiment in HCC827 cells containing EGFR deletion mutant.

FIG. 10 depicts the results of compound I-7 in a “washout” experiment ascompared to results of an EGF control in A431 cells containing EGFR wildtype.

FIG. 11 depicts MS analysis confirming covalent modification of JAK-3kinase at Cys909 by compound I-7.

FIG. 12 depicts dose-response inhibition of P-Stat5 with compound I-2 inIL-2 stimulated CTLL-2 cells; and dose-response inhibition of P-JAK-3with compound I-2 in IL-2 stimulated CTLL-2 cells.

FIG. 13 depicts dose-response inhibition of P-Stat5 with compound I-4 inIL-2 stimulated CTLL-2 cells; and dose-response inhibition of P-JAK-3with compound I-4 in IL-2 stimulated CTLL-2 cells.

FIG. 14 depicts dose-response inhibition of P-Stat5 with compound I-7 inIL-2 stimulated CTLL-2 cells.

FIG. 15 depicts MS analysis confirming covalent modification of BTK bycompound I-7.

FIG. 16 depicts a Western blot showing BTK protein available to theprobe compound I-215 after treating with varying amounts of I-7.

FIG. 17 depicts quantitation of the Western blot results in FIG. 16.

FIG. 18 depicts a Western blot for a washout experiment with compoundI-7 and probe compound I-215.

FIG. 19 depicts quantitation of the Western blot results in FIG. 18.

FIG. 20 depicts an amino acid sequence for BTK (SEQ ID 1).

FIG. 21 depicts an amino acid sequence for TEC (SEQ ID 2).

FIG. 22 depicts an amino acid sequence for ITK (SEQ ID 3).

FIG. 23 depicts an amino acid sequence for BMX (SEQ ID 4).

FIG. 24 depicts an amino acid sequence for TXK (SEQ ID 5).

FIG. 25 depicts an amino acid sequence for JAK3 (SEQ ID 6).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description ofCompounds of the Invention

In certain embodiments, the present invention provides a compound offormula I-a or I-b:

-   or a pharmaceutically acceptable salt thereof, wherein:-   Ring A is an optionally substituted group selected from phenyl, a    3-7 membered saturated or partially unsaturated carbocyclic ring, an    8-10 membered bicyclic saturated, partially unsaturated or aryl    ring, a 5-6 membered monocyclic heteroaryl ring having 1-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    a 4-7 membered saturated or partially unsaturated heterocyclic ring    having 1-3 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, a 7-10 membered bicyclic saturated or partially    unsaturated heterocyclic ring having 1-5 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or an 8-10 membered    bicyclic heteroaryl ring having 1-5 heteroatoms independently    selected from nitrogen, oxygen, or sulfur;-   Ring B is an optionally substituted group selected from phenyl, a    3-7 membered saturated or partially unsaturated carbocyclic ring, an    8-10 membered bicyclic saturated, partially unsaturated or aryl    ring, a 5-6 membered monocyclic heteroaryl ring having 1-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    a 4-7 membered saturated or partially unsaturated heterocyclic ring    having 1-3 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, a 7-10 membered bicyclic saturated or partially    unsaturated heterocyclic ring having 1-5 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or an 8-10 membered    bicyclic heteroaryl ring having 1-5 heteroatoms independently    selected from nitrogen, oxygen, or sulfur;-   R¹ is a warhead group;-   R^(y) is hydrogen, halogen, —CN, —CF₃, C₁₋₄ aliphatic, C₁₋₄    haloaliphatic, —OR, —C(O)R, or —C(O)N(R)₂;-   each R group is independently hydrogen or an optionally substituted    group selected from C₁ aliphatic, phenyl, a 4-7 membered heterocylic    ring having 1-2 heteroatoms independently selected from nitrogen,    oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur;-   W¹ and W² are each independently a covalent bond or a bivalent C₁₋₃    alkylene chain wherein one methylene unit of W¹ or W² is optionally    replaced by —NR²—, —N(R²)C(O)—, —C(O)N(R²)—, —N(R²)SO₂—, —SO₂N(R²)—,    —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO— or —SO₂—;-   R² is hydrogen, optionally substituted C₁₋₆ aliphatic, or —C(O)R,    or:    -   R² and a substituent on Ring A are taken together with their        intervening atoms to form a 4-6 membered saturated, partially        unsaturated, or aromatic fused ring, or:    -   R² and R^(y) are taken together with their intervening atoms to        form a 4-7 membered partially unsaturated or aromatic fused        ring;-   m and p are independently 0-4; and-   R^(x) and R^(v) are independently selected from —R, halogen, —OR,    —O(CH₂)_(q)OR, —CN, —NO₂, —SO₂R, —SO₂N(R)₂, —SOR, —C(O)R, —CO₂R,    —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂, —NRSO₂R, or —N(R)₂, wherein q is    1-4; or:    -   R^(x) and R¹ when concurrently present on Ring B are taken        together with their intervening atoms to form a 5-7 membered        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, wherein said ring is substituted with a warhead group        and 0-3 groups independently selected from oxo, halogen, —CN, or        C₁₋₆ aliphatic; or    -   R^(v) and R¹ when concurrently present on Ring A are taken        together with their intervening atoms to form a 5-7 membered        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, wherein said ring is substituted with a warhead group        and 0-3 groups independently selected from oxo, halogen, —CN, or        C₁₋₆ aliphatic.

2. Compounds and Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₆ hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.Suitable aliphatic groups include, but are not limited to, linear orbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₈ (or C₁₋₆) saturated orunsaturated, straight or branched, hydrocarbon chain”, refers tobivalent alkylene, alkenylene, and alkynylene chains that are straightor branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

As used herein, the term “cyclopropylenyl” refers to a bivalentcyclopropyl group of the following structure:

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic andbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains three to seven ring members. The term “aryl”may be used interchangeably with the term “aryl ring”. In certainembodiments of the present invention, “aryl” refers to an aromatic ringsystem which includes, but not limited to, phenyl, biphenyl, naphthyl,anthracyl and the like, which may bear one or more substituents. Alsoincluded within the scope of the term “aryl”, as it is used herein, is agroup in which an aromatic ring is fused to one or more non-aromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-”, used alone or as part of alarger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14 nt electrons shared in a cyclic array; and having,in addition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring”, “heteroarylgroup”, or “heteroaromatic”, any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclicradical”, and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclicgroup”, “heterocyclic moiety”, and “heterocyclic radical”, are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(◯); —(CH₂)₀₋₄OR^(◯); —O(CH₂)₀₋₄R^(◯), —O—(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄CH(OR^(◯))₂; —(CH₂)₀₋₄SR^(◯); —(CH₂)₀₋₄Ph, which may besubstituted with R^(◯); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(◯); —CH═CHPh, which may be substituted with R^(◯);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(◯); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(◯))₂; —(CH₂)₀₋₄N(R^(◯))C(O)R^(◯);—N(R^(◯))C(S)R^(◯); —(CH₂)₀₋₄N(R)C(O)NR^(◯) ₂; —N(R^(◯))C(S)NR^(◯) ₂;—(CH₂)₀₋₄N(R^(◯))C(O)OR^(◯); —N(R^(◯))N(R^(◯))C(O)R^(◯);—N(R^(◯))N(R^(◯))C(O)NR^(◯) ₂; —N(R^(◯))N(R^(◯))C(O)OR^(◯);—(CH₂)₀₋₄C(O)R^(◯); —C(S)R^(◯); —(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄C(O)SR^(◯); —(CH₂)₀₋₄C(O)OSiR^(◯) ₃; —(CH₂)₀₋₄OC(O)R^(◯);—OC(O)(CH₂)₀₋₄SR^(◯), SC(S)SR^(◯); —(CH₂)₀₋₄SC(O)R^(◯);—(CH₂)₀₋₄C(O)NR^(◯) ₂; —C(S)NR^(◯) ₂; —C(S)SR^(◯); —SC(S)SR^(◯),—(CH₂)₀₋₄OC(O)NR^(◯) ₂; —C(O)N(OR^(◯))R^(◯); —C(O)C(O)R^(◯);—C(O)CH₂C(O)R^(◯); —C(NOR^(◯))R^(◯); —(CH₂)₀₋₄SSR^(◯);—(CH₂)₀₋₄S(O)₂R^(◯); —(CH₂)₀₋₄S(O)₂OR^(◯); —(CH₂)₀₋₄OS(O)₂R^(◯);—S(O)₂NR^(◯) ₂; —(CH₂)₀₋₄S(O)R^(◯); —N(R^(◯))S(O)₂NR^(◯) ₂;—N(R^(◯))S(O)₂R^(◯); —N(OR^(◯))R^(◯); —C(NH)NR^(◯) ₂; —P(O)₂R^(◯);—P(O)R^(◯) ₂; —OP(O)R^(◯) ₂; —OP(O)(OR^(◯))₂; SiR^(◯) ₃; —(C₁₋₄ straightor branched alkylene)O—N(R^(◯))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(◯))₂, wherein each R^(◯) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(◯), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(◯) (or the ring formed by takingtwo independent occurrences of R^(◯) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(◯) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R)S(O)₂R^(†); wherein each R^(†) isindependently hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention. In someembodiments, the R¹ group of formula I-a and I-b comprises one or moredeuterium atoms.

As used herein, the term “irreversible” or “irreversible inhibitor”refers to an inhibitor (i.e. a compound) that is able to be covalentlybonded to a target protein kinase in a substantially non-reversiblemanner. That is, whereas a reversible inhibitor is able to bind to (butis generally unable to form a covalent bond) the target protein kinase,and therefore can become dissociated from the target protein kinase, anirreversible inhibitor will remain substantially bound to the targetprotein kinase once covalent bond formation has occurred. Irreversibleinhibitors usually display time dependency, whereby the degree ofinhibition increases with the time with which the inhibitor is incontact with the enzyme. Methods for identifying if a compound is actingas an irreversible inhibitor are known to one of ordinary skill in theart. Such methods include, but are not limited to, enzyme kineticanalysis of the inhibition profile of the compound with the proteinkinase target, the use of mass spectrometry of the protein drug targetmodified in the presence of the inhibitor compound, discontinuousexposure, also known as “washout,” experiments, and the use of labeling,such as radiolabelled inhibitor, to show covalent modification of theenzyme, as well as other methods known to one of skill in the art.

One of ordinary skill in the art will recognize that certain reactivefunctional groups can act as “warheads.” As used herein, the term“warhead” or “warhead group” refers to a functional group present on acompound of the present invention wherein that functional group iscapable of covalently binding to an amino acid residue (such ascysteine, lysine, histidine, or other residues capable of beingcovalently modified) present in the binding pocket of the targetprotein, thereby irreversibly inhibiting the protein. It will beappreciated that the -L-Y group, as defined and described herein,provides such warhead groups for covalently, and irreversibly,inhibiting the protein.

As used herein, the term “inhibitor” is defined as a compound that bindsto and/or inhibits the target protein kinase with measurable affinity.In certain embodiments, an inhibitor has an IC₅₀ and/or binding constantof less about 50 μM, less than about 1 μM, less than about 500 nM, lessthan about 100 nM, or less than about 10 nM.

The terms “measurable affinity” and “measurably inhibit,” as usedherein, means a measurable change in at least one of ErbB1, ErbB2,ErbB3, ErbB4, a TEC-kinase, and/or JAK3 activity between a samplecomprising a compound of the present invention, or composition thereof,and at least one of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/orJAK3, and an equivalent sample comprising at least one of ErbB1, ErbB2,ErbB3, ErbB4, a TEC-kinase, and/or JAK3, in the absence of saidcompound, or composition thereof.

3. Description of Exemplary Compounds

According to one aspect, the present invention provides a compound offormula I-a or I-b,

-   or a pharmaceutically acceptable salt thereof, wherein:-   Ring A is an optionally substituted group selected from phenyl, a    3-7 membered saturated or partially unsaturated carbocyclic ring, an    8-10 membered bicyclic saturated, partially unsaturated or aryl    ring, a 5-6 membered monocyclic heteroaryl ring having 1-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    a 4-7 membered saturated or partially unsaturated heterocyclic ring    having 1-3 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, a 7-10 membered bicyclic saturated or partially    unsaturated heterocyclic ring having 1-5 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or an 8-10 membered    bicyclic heteroaryl ring having 1-5 heteroatoms independently    selected from nitrogen, oxygen, or sulfur;-   Ring B is an optionally substituted group selected from phenyl, a    3-7 membered saturated or partially unsaturated carbocyclic ring, an    8-10 membered bicyclic saturated, partially unsaturated or aryl    ring, a 5-6 membered monocyclic heteroaryl ring having 1-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    a 4-7 membered saturated or partially unsaturated heterocyclic ring    having 1-3 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, a 7-10 membered bicyclic saturated or partially    unsaturated heterocyclic ring having 1-5 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or an 8-10 membered    bicyclic heteroaryl ring having 1-5 heteroatoms independently    selected from nitrogen, oxygen, or sulfur;-   R¹ is -L-Y, wherein:    -   L is a covalent bond or a bivalent C₁₋₈ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by cyclopropylene, —NR—, —N(R)C(O)—,        —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—,        —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with at 1-4 groups        independently selected from -Q-Z, oxo, NO₂, halogen, CN, or C₁₋₆        aliphatic, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —NR—, —S—, —O—, —C(O)—, —SO—, or            —SO₂—; and        -   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with            oxo, halogen, or CN;-   R^(y) is hydrogen, halogen, —CN, —CF₃, C₁₋₄ aliphatic, C₁₋₄    haloaliphatic, —OR, —C(O)R, or —C(O)N(R)₂;-   each R group is independently hydrogen or an optionally substituted    group selected from C₁-aliphatic, phenyl, a 4-7 membered heterocylic    ring having 1-2 heteroatoms independently selected from nitrogen,    oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur;-   W¹ and W² are each independently a covalent bond or a bivalent C₁₋₃    alkylene chain wherein one methylene unit of W¹ or W² is optionally    replaced by —NR²—, —N(R²)C(O)—, —C(O)N(R²)—, —N(R²)SO₂—, —SO₂N(R²)—,    —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO— or —SO₂—;-   R² is hydrogen, optionally substituted C₁₋₆ aliphatic, or —C(O)R,    or:    -   R² and a substituent on Ring A are taken together with their        intervening atoms to form a 4-6 membered partially unsaturated        or aromatic fused ring; or    -   R² and R^(y) are taken together with their intervening atoms to        form a 4-6 membered saturated, partially unsaturated, or        aromatic fused ring;-   m and p are independently 0-4; and-   R^(x) and R^(v) are independently selected from —R, halogen, —OR,    —O(CH₂)_(q)OR, —CN, —NO₂, —SO₂R, —SO₂N(R)₂, —SOR, —C(O)R, —CO₂R,    —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂, —NRSO₂R, or —N(R)₂, or:    -   R^(x) and R¹ when concurrently present on Ring B are taken        together with their intervening atoms to form a 5-7 membered        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, wherein said ring is substituted with a warhead group        and 0-3 groups independently selected from oxo, halogen, —CN, or        C₁₋₆ aliphatic; or    -   R^(v) and R¹ when concurrently present on Ring A are taken        together with their intervening atoms to form a 5-7 membered        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, wherein said ring is substituted with a warhead group        and 0-3 groups independently selected from oxo, halogen, —CN, or        C₁₋₆ aliphatic.

As defined generally above, Ring A is an optionally substituted groupselected from phenyl, a 3-7 membered saturated or partially unsaturatedcarbocyclic ring, an 8-10 membered bicyclic saturated, partiallyunsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, a 4-7 membered saturated or partially unsaturated heterocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur, a 7-10 membered bicyclic saturated or partiallyunsaturated heterocyclic ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclicheteroaryl ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, Ring A is anoptionally substituted phenyl group. In some embodiments, Ring A is anoptionally substituted naphthyl ring or a bicyclic 8-10 memberedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain other embodiments, Ring A is anoptionally substituted 3-7 membered carbocyclic ring. In yet otherembodiments, Ring A is an optionally substituted 4-7 memberedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In certain embodiments, Ring A is substituted as defined herein. In someembodiments, Ring A is substituted with one, two, or three groupsindependently selected from halogen, R^(◯), or —(CH₂)₀₋₄₀OR^(◯), or—O(CH₂)₀₋₄R^(◯), wherein each R^(◯) is as defined herein. Exemplarysubstituents on Ring A include Br, I, Cl, methyl, —CF₃, —C≡CH,—OCH₂phenyl, —OCH₂(fluorophenyl), or —OCH₂pyridyl.

Exemplary Ring A groups are set forth in Table 1.

TABLE 1 Exemplary Ring A Groups

i

ii

iii

iv

v

vi

vii

viii

ix

x

xi

xii

xiii

xiv

xv

xvi

xvii

xviii

xix

xx

xxi

xxii

xxiii

xxiv

xxv

xxvi

xxvii

xxviii

xxix

xxx

xxxi

xxxii

xxxiii

xxxiv

xxxv

xxxvi

xxxvii

xxxviii

xxxix

xl

xli

xlii

xliii

xliv

xlv

xlvi

xlvii

xlviii

xlix

l

li

lii

liii

liv

lv

lvi

lvii

lviii

lix

lx

lxi

lxii

lxiii

lxiv

lxv

lxvi

lxvii

lxviii

lxix

lxx

lxxi

lxxii

lxxiii

lxxiv

lxxv

lxxvi

lxxvii

lxxviii

lxxix

lxxx

lxxxi

lxxxii

lxxxiii

lxxxiv

lxxxvwherein each R^(◯), R^(†), and R¹ is as defined above and described inclasses and subclasses herein.

In certain embodiments, Ring A is selected from i, ii, iv, v, vi, vii,ix, xiv, xvi, lii, lxiii, lxxi, lxxiv, lxxvi, lxxviii, and lxxxi.

As defined generally above, Ring B is an optionally substituted groupselected from phenyl, a 3-7 membered saturated or partially unsaturatedcarbocyclic ring, an 8-10 membered bicyclic saturated, partiallyunsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, a 4-7 membered saturated or partially unsaturated heterocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur, a 7-10 membered bicyclic saturated or partiallyunsaturated heterocyclic ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclicheteroaryl ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, Ring B is anoptionally substituted phenyl group. In some embodiments, Ring B is anoptionally substituted naphthyl ring or a bicyclic 8-10 memberedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain other embodiments, Ring B is anoptionally substituted 3-7 membered carbocyclic ring. In yet otherembodiments, Ring B is an optionally substituted 4-7 memberedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, Ring B is phenyl. In some embodiments, Ring B is a6-membered heteroaryl ring having 1-3 nitrogens. In some embodiments,Ring B is a 5-membered heteroaryl ring having 1 or 2 or 3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, Ring B is a 5-6 membered saturated heterocyclicring having 1 nitrogen. In some embodiments, Ring B is a 9-10 memberedbicyclic partially saturated heteroaryl ring having 1-3 nitrogens. Insome embodiments, Ring B is a 9-10 membered bicyclic partially saturatedheteroaryl ring having 1 nitrogen. In some embodiments, Ring B is a 9-10membered bicyclic partially saturated heteroaryl ring having 1 nitrogenand 1 oxygen.

In some embodiments, Ring B is an optionally substituted group selectedfrom phenyl, pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, pyrrolidinyl,piperdinyl, indolinyl, indazolyl, and isoindolinyl.

Exemplary Ring B groups are set forth in Table 2.

TABLE 2 Ring B Groups

i

ii

iii

iv

v

vi

vii

viii

ix

x

xi

xii

xiii

xiv

xv

xvi

xvii

xviii

xix

xx

xxi

xxii

xxiii

xxiv

xxv

xxvi

xxvii

xxviii

xxix

xxx

xxxi

xxxii

xxxiii

xxxiv

xxxv

xxxvi

xxxvii

xxxviii

xxxix

xl

xli

xlii

xliii

xliv

xlv

xlvi

xlvii

xlviii

xlix

l

li

lii

liii

liv

lv

lvi

lvii

lviii

lix

lx

lxi

lxii

lxiii

lxiv

lxv

lxvi

lxvii

lxviii

lxix

lxx

lxxi

lxxii

lxxiii

lxxiv

lxxv

lxxvi

lxxvii

lxxviii

lxxix

lxxx

lxxxi

lxxxii

lxxxiii

lxxxiv

lxxxv

lxxxvi

lxxxvii

lxxxviii

lxxxix

xc

xci

xcii

xciii

xciv

xcv

xcvi

xcvii

xcviii

xcix

c

ci

cii

ciii

civ

cv

cvi

cvii

cviii

cix

cx

cxi

cxii

cxiii

cxiv

cxv

cxvi

cxvii

cxviii

cxxix

cxx

cxxi

cxxiiwherein each R¹ and R^(x) is as defined above and described in classesand subclasses herein.

In certain embodiments, Ring B is selected from i, ii, iii, iv, v, ix,x, xi, xiii, xvi, xvii, xix, xx, xxv, xxvi, xxxii, xxxiv, xxxv, xxxviii,xlii, xlvi, xlviii, l, lviii, lxiv, lxxviii, lxxxiii, lxxxvi, xciv, c,ci, cii, ciii, civ, and cv.

In some embodiments, the m moiety of formula I is 1, 2, 3 or 4. In someembodiments, m is 1. In other embodiments, m is 0.

In some embodiments, the p moiety of formula I is 1, 2, 3 or 4. In someembodiments, p is 1. In other embodiments, p is 0.

As defined generally above, each R^(x) group of formula I isindependently selected from —R, halogen, —OR, —O(CH₂)_(q)OR, —CN, —NO₂,—SO₂R, —SO₂N(R)₂, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂,—NRSO₂R, or —N(R)₂, wherein q is 1-4, or R^(x) and R¹ when concurrentlypresent on Ring B are taken together with their intervening atoms toform a 5-7 membered saturated, partially unsaturated, or aryl ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, wherein said ring is substituted with a warhead group and 0-3groups independently selected from oxo, halogen, CN, or C₁₋₆ aliphatic.

In some embodiments, each instance of R^(x) is independently selectedfrom —R, —OR, —O(CH₂)_(q)OR, or halogen. In certain embodiments, R^(x)is lower alkyl, lower alkoxy, lower alkoxyalkoxy, or halogen. ExemplaryR^(x) groups include methyl, methoxy, methoxyethoxy and fluoro. In someembodiments, R^(x) is hydrogen.

As defined generally above, each R^(v) group of formula I isindependently selected from —R, halogen, —OR, —O(CH₂)_(q)OR, —CN, —NO₂,—SO₂R, —SO₂N(R)₂, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂,—NRSO₂R, or —N(R)₂, wherein q is 1-4, or R^(v) and R¹ when concurrentlypresent on Ring A are taken together with their intervening atoms toform a 5-7 membered saturated, partially unsaturated, or aryl ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, wherein said ring is substituted with a warhead group and 0-3groups independently selected from oxo, halogen, CN, or C₁₋₆ aliphatic.

In some embodiments, each instance of R^(v) is independently selectedfrom —R, —OR, —O(CH₂)_(q)OR, or halogen. In certain embodiments, R^(v)is lower alkyl, lower alkoxy, lower alkoxyalkoxy, or halogen. ExemplaryR^(v) groups include methyl, methoxy, trifluoromethyl, methoxyethoxy,and chloro. In some embodiments, R^(v) is hydrogen.

In some embodiments, the q moiety is 1, 2, 3, or 4. In certainembodiments, q is 1. In certain other embodiments, q is 2.

As defined generally above, R^(y) is hydrogen, halogen, —CN, —CF₃, C₁₋₄aliphatic, C₁₋₄ haloaliphatic, —OR, —C(O)R, or —C(O)N(R)₂, where R is asdefined above and described herein. In certain embodiments, R^(y) ishydrogen, halogen, —CN, —CF₃, lower alkyl or lower haloalkyl, —C≡CR andcyclopropyl. In other embodiments, R^(y) is —OR, —C(O)R, or —C(O)N(R)₂.In certain embodiments, R^(y) is —OCH₃. In certain other embodiments,R^(y) is —C(O)CH₃. In yet other embodiments, R^(y) is —C(O)NHR. In someembodiments, R^(y) is hydrogen. In certain embodiments, R^(y) isfluorine. In certain other embodiments, R^(y) is methyl.

As generally defined above, W¹ and W² are each independently a covalentbond or a bivalent C₁₋₃ alkylene chain wherein one methylene unit of W¹or W² is optionally replaced by —NR²—, —N(R²)C(O)—, —C(O)N(R²)—,—N(R²)SO₂—, —SO₂N(R²)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO— or—SO₂—. In certain embodiments, W¹ and W² are the same. In someembodiments, W¹ and W² are different.

In some embodiments, W¹ is a covalent bond. In certain embodiments, W¹is a bivalent C₁₋₃ alkylene chain wherein one methylene unit of W¹ isoptionally replaced by —NR²—, —N(R²)C(O)—, —C(O)N(R²)—, —N(R²)SO₂—,—SO₂N(R²)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO— or —SO₂—. Incertain embodiments, W¹ is —C(═O), —NR²—, —S—, or —O—. In someembodiments, W¹ is —NR²—. In other embodiments, W¹ is —O—. In certainembodiments, W¹ is —NH—, —S—, or —O—. In some embodiments, W¹ is —CH₂O—,—CH₂S—, or —CH₂NH—. In some aspects, W¹ is —OCH₂—, —SCH₂—, —NHCH₂—, or—CH₂CH₂—.

In certain embodiments, W² is a covalent bond. In some embodiments, W²is a bivalent C₁₋₃ alkylene chain wherein one methylene unit of W² isoptionally replaced by —NR²—, —N(R²)C(O)—, —C(O)N(R²)—, —N(R²)SO₂—,—SO₂N(R²)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO— or —SO₂—. Incertain embodiments, W² is —C(═O), —NR²—, —S—, or —O—. In someembodiments, W² is —NR²—. In other embodiments, W² is —O—. In certainembodiments, W² is —NH—, —S—, or —O—. In some embodiments, W² is —CH₂O—,—CH₂S—, or —CH₂NH—. In some aspects, W² is —OCH₂—, —SCH₂—, —NHCH₂—, or—CH₂CH₂—.

In some embodiments, Ring B is phenyl, thus forming a compound offormula II-a or II-b:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,m, p, R^(x), R^(y), R^(v), W¹, W², and R¹ is as defined above anddescribed in classes and subclasses above and herein.

In certain embodiments, Ring A is phenyl, thus forming a compound offormula III-a or III-b:

or a pharmaceutically acceptable salt thereof, wherein each of Ring B,m, p, R^(x), R^(y), R^(v), W¹, W², and R¹ is as defined above anddescribed in classes and subclasses above and herein.

In certain embodiments, Ring A is phenyl and Ring B is phenyl, thusforming a compound of formula IV-a or IV-b:

or a pharmaceutically acceptable salt thereof, wherein each of m, p,R^(x), R^(y), R^(v), W¹, W², and R¹ is as defined above and described inclasses and subclasses above and herein.

As defined generally above, each R² is independently hydrogen,optionally substituted C₁₋₆ aliphatic, or —C(O)R, or R² and asubstituent on Ring A are taken together with their intervening atoms toform a 4-6 membered partially unsaturated or aromatic fused ring, or R²and R^(y) are taken together with their intervening atoms to form a 4-6membered saturated, partially unsaturated, or aromatic fused ring.According to one aspect, R² is hydrogen. According to another aspect, R²is —C(O)R, wherein R is an optionally substituted C₁₋₆ aliphatic group.

According to some aspects, R² and a substituent on Ring A are takentogether with their intervening atoms to form a 4-7 membered saturatedor partially unsaturated ring, thus forming a compound of formula I-a-ior I-b-i:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R^(x), and m are as defined above and described in classes andsubclasses above and herein.

Similar to the formation of compounds of formulae I-a-i and I-b-i above,it will be understood by one skilled in the art that compounds offormulae II-a, II-b, III-a, III-b, IV-a, and IV-b, will formcorresponding compounds II-a-i, II-b-i, III-a-i, III-b-i, IV-a-i, andIV-b-i when R² and a substituent on Ring A are taken together with theirintervening atoms to form a 4-7 membered saturated or partiallyunsaturated ring.

According to some aspects, R² and R^(y) are taken together with theirintervening atoms to form a 4-7 partially unsaturated ring, thus forminga compound of formula I-a-ii or I-b-ii:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R^(x), and m are as defined above and described in classes andsubclasses above and herein.

Similar to the formation of compounds of formulae I-a-ii and I-b-iiabove, it will be understood by one skilled in the art that compounds offormulae II-a, II-b, III-a, III-b, IV-a, and IV-b, will formcorresponding compounds II-a-ii, II-b-ii, III-a-ii, III-b-ii, IV-a-ii,and IV-b-ii when R² and R^(y) are taken together with their interveningatoms to form a 4-7 membered partially unsaturated ring.

As defined generally above, the R¹ group of formulae I and II is -L-Y,wherein:

-   -   L is a covalent bond or a bivalent C₁₋₈ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by cyclopropylene, —NR—, —N(R)C(O)—,        —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—,        —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with 1-4 R^(e)        groups; and    -   each R^(e) is independently selected from -Q-Z, oxo, NO₂,        halogen, CN, a suitable leaving group, or a C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—,            —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—,            or —SO₂N(R)—; and    -   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN.

In certain embodiments, L is a covalent bond.

In certain embodiments, L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain. In certain embodiments, L is—CH₂—.

In certain embodiments, L is a covalent bond, —CH₂—, —NH—, —CH₂NH—,—NHCH₂—, —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,—NHC(O)CH₂OC(O)—, or —SO₂NH—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and one or twoadditional methylene units of L are optionally and independentlyreplaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—,—SO₂—, —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,—N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and oneor two additional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, and one or two additionalmethylene units of L are optionally and independently replaced bycyclopropylene, —O—, —N(R)—, or —C(O)—.

As described above, in certain embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onedouble bond. One of ordinary skill in the art will recognize that such adouble bond may exist within the hydrocarbon chain backbone or may be“exo” to the backbone chain and thus forming an alkylidene group. By wayof example, such an L group having an alkylidene branched chain includes—CH₂C(═CH₂)CH₂—. Thus, in some embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onealkylidenyl double bond. Exemplary L groups include —NHC(O)C(═CH₂)CH₂—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—. In certain embodiments, Lis —C(O)CH═CH(CH₃)—, —C(O)CH═CHCH₂NH(CH₃)—, —C(O)CH═CH(CH₃)—,—C(O)CH═CH—, —CH₂C(O)CH═CH—, —CH₂C(O)CH═CH(CH₃)—, —CH₂CH₂C(O)CH═CH—,—CH₂CH₂C(O)CH═CHCH₂—, —CH₂CH₂C(O)CH═CHCH₂NH(CH₃)—, or—CH₂CH₂C(O)CH═CH(CH₃)—, or —CH(CH₃)OC(O)CH═CH—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —OC(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—,—SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and one or twoadditional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—. In some embodiments,L is —CH₂OC(O)CH═CHCH₂—, —CH₂—OC(O)CH═CH—, or —CH(CH═CH₂)OC(O)CH═CH—.

In certain embodiments, L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,—NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,—NRC(O)(C═N₂)C(O)—, —NRC(O)CH═CHCH₂N(CH₃)—, —NRSO₂CH═CH—,—NRSO₂CH═CHCH₂—, —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,—CH₂NRC(O)CH═CH—, —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-, whereineach R is independently hydrogen or optionally substituted C₁₋₆aliphatic.

In certain embodiments, L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,—NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,—NHC(O)(C═N₂)C(O)—, —NHC(O)CH═CHCH₂N(CH₃)—, —NHSO₂CH═CH—,—NHSO₂CH═CHCH₂—, —NHC(O)CH═CHCH₂O—, —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—,—CH₂NHC(O)CH═CH—, —CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one triple bond. In certainembodiments, L is a bivalent C₂₋₈ straight or branched, hydrocarbonchain wherein L has at least one triple bond and one or two additionalmethylene units of L are optionally and independently replaced by—NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—,or —C(O)—. In some embodiments, L has at least one triple bond and atleast one methylene unit of L is replaced by —N(R)—, —N(R)C(O)—, —C(O)—,—C(O)O—, or —OC(O)—, or —O—.

Exemplary L groups include —C≡C—, —C≡CCH₂N(isopropyl)-,—NHC(O)C≡CCH₂CH₂—, —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or—CH₂OC(═O)C≡C—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein one methylene unit of L is replaced bycyclopropylene and one or two additional methylene units of L areindependently replaced by —C(O)—, —NRC(O)—, —C(O)NR—, —N(R)SO₂—, or—SO₂N(R)—. Exemplary L groups include —NHC(O)-cyclopropylene-SO₂— and—NHC(O)— cyclopropylene-.

As defined generally above, Y is hydrogen, C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN, or a 3-10 membered monocyclicor bicyclic, saturated, partially unsaturated, or aryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, andwherein said ring is substituted with at 1-4 R^(e) groups, each R^(e) isindependently selected from -Q-Z, oxo, NO₂, halogen, CN, a suitableleaving group, or C₁₋₆ aliphatic, wherein Q is a covalent bond or abivalent C₁₋₆ saturated or unsaturated, straight or branched,hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —N(R)—, —S—, —O—, —C(O)—,—OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, or—SO₂N(R)—; and, Z is hydrogen or C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN.

In certain embodiments, Y is hydrogen.

In certain embodiments, Y is C₁₋₆ aliphatic optionally substituted withoxo, halogen, NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN. In otherembodiments, Y is C₂₋₆ alkynyl optionally substituted with oxo, halogen,NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyl. In otherembodiments, Y is C₂₋₄ alkynyl.

In other embodiments, Y is C₁₋₆ alkyl substituted with oxo, halogen,NO₂, or CN. Such Y groups include —CH₂F, —CH₂Cl, —CH₂CN, and —CH₂NO₂.

In certain embodiments, Y is a saturated 3-6 membered monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, wherein Y is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein.

In some embodiments, Y is a saturated 3-4 membered heterocyclic ringhaving 1 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-2 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Exemplary such rings are epoxide and oxetanerings, wherein each ring is substituted with 1-2 R^(e) groups, whereineach R^(e) is as defined above and described herein.

In other embodiments, Y is a saturated 5-6 membered heterocyclic ringhaving 1-2 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-4 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Such rings include piperidine andpyrrolidine, wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R, Q, Z, and R^(e) is as defined above and describedherein.

In some embodiments, Y is a saturated 3-6 membered carbocyclic ring,wherein said ring is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein. In certain embodiments,Y is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein. In certain embodiments, Y is

R^(e) is as defined above and described herein. In certain embodiments,Y is cyclopropyl optionally substituted with halogen, CN or NO₂.

In certain embodiments, Y is a partially unsaturated 3-6 memberedmonocyclic ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein.

In some embodiments, Y is a partially unsaturated 3-6 memberedcarbocyclic ring, wherein said ring is substituted with 1-4 R^(e)groups, wherein each R^(e) is as defined above and described herein. Insome embodiments, Y is cyclopropenyl, cyclobutenyl, cyclopentenyl, orcyclohexenyl wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R^(e) is as defined above and described herein.

In certain embodiments, Y is a partially unsaturated 4-6 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein. In certain embodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is a 6-membered aromatic ring having 0-2nitrogens wherein said ring is substituted with 1-4 R^(e) groups,wherein each R^(e) group is as defined above and described herein. Incertain embodiments, Y is phenyl, pyridyl, or pyrimidinyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein.

In some embodiments, Y is selected from:

wherein each R^(e) is as defined above and described herein.

In other embodiments, Y is a 5-membered heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein said ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In someembodiments, Y is a 5 membered partially unsaturated or aryl ring having1-3 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein said ring is substituted with 1-4 R^(e) groups, whereineach R^(e) group is as defined above and described herein. Exemplarysuch rings are isoxazolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,pyrrolyl, furanyl, thienyl, triazole, thiadiazole, and oxadiazole,wherein each ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In certainembodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is an 8-10 membered bicyclic, saturated,partially unsaturated, or aryl ring having 0-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein said ring issubstituted with 1-4 R^(e) groups, wherein R^(e) is as defined above anddescribed herein. According to another aspect, Y is a 9-10 memberedbicyclic, partially unsaturated, or aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidring is substituted with 1-4 R^(e) groups, wherein R^(e) is as definedabove and described herein. Exemplary such bicyclic rings include2,3-dihydrobenzo[d]isothiazole, wherein said ring is substituted with1-4 R^(e) groups, wherein R^(e) is as defined above and describedherein.

As defined generally above, each R^(e) group is independently selectedfrom -Q-Z, oxo, NO₂, halogen, CN, a suitable leaving group, or C₁₋₆aliphatic optionally substituted with oxo, halogen, NO₂, or CN, whereinQ is a covalent bond or a bivalent C₁₋₆ saturated or unsaturated,straight or branched, hydrocarbon chain, wherein one or two methyleneunits of Q are optionally and independently replaced by —N(R)—, —S—,—O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—,—N(R)SO₂—, or —SO₂N(R)—; and Z is hydrogen or C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN.

In certain embodiments, R^(e) is C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN. In other embodiments, R^(e) is oxo, NO₂,halogen, or CN.

In some embodiments, R^(e) is -Q-Z, wherein Q is a covalent bond and Zis hydrogen (i.e., R^(e) is hydrogen). In other embodiments, R^(e) is-Q-Z, wherein Q is a bivalent C₁₋₆ saturated or unsaturated, straight orbranched, hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —NR—, —NRC(O)—, —C(O)NR—, —S—,—O—, —C(O)—, —SO—, or —SO₂—. In other embodiments, Q is a bivalent C₂₋₆straight or branched, hydrocarbon chain having at least one double bond,wherein one or two methylene units of Q are optionally and independentlyreplaced by —NR—, —NRC(O)—, —C(O)NR—, —S—, —O—, —C(O)—, —SO—, or —SO₂—.In certain embodiments, the Z moiety of the R^(e) group is hydrogen. Insome embodiments, -Q-Z is —NHC(O)CH═CH₂ or —C(O)CH═CH₂.

In certain embodiments, each R^(e) is independently selected from fromoxo, NO₂, CN, fluoro, chloro, —NHC(O)CH═CH₂, —C(O)CH═CH₂, —CH₂CH═CH₂,—C≡CH, —C(O)OCH₂Cl, —C(O)OCH₂F, —C(O)OCH₂CN, —C(O)CH₂Cl, —C(O)CH₂F,—C(O)CH₂CN, or —CH₂C(O)CH₃.

In certain embodiments, R^(e) is a suitable leaving group, ie a groupthat is subject to nucleophilic displacement. A “suitable leaving” is achemical group that is readily displaced by a desired incoming chemicalmoiety such as the thiol moiety of a cysteine of interest. Suitableleaving groups are well known in the art, e.g., see, “Advanced OrganicChemistry,” Jerry March, 5^(th) Ed., pp. 351-357, John Wiley and Sons,N.Y. Such leaving groups include, but are not limited to, halogen,alkoxy, sulphonyloxy, optionally substituted alkylsulphonyloxy,optionally substituted alkenylsulfonyloxy, optionally substitutedarylsulfonyloxy, acyl, and diazonium moieties. Examples of suitableleaving groups include chloro, iodo, bromo, fluoro, acetoxy,methanesulfonyloxy (mesyloxy), tosyloxy, triflyloxy,nitro-phenylsulfonyloxy (nosyloxy), and bromo-phenylsulfonyloxy(brosyloxy).

In certain embodiments, the following embodiments and combinations of-L-Y apply:

-   -   (a) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (b) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,        —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—,        and one or two additional methylene units of L are optionally        and independently replaced by cyclopropylene, —O—, —N(R)—, or        —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (c) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, and one or two        additional methylene units of L are optionally and independently        replaced by cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y is        hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (d) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (e) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —OC(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (f) L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,        —NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—,        —NRSO₂CH═CHCH₂—, —NRC(O)(C═N₂)—, —NRC(O)(C═N₂)C(O)—,        —NRC(O)CH═CHCH₂N(CH₃)—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,        —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,        —CH₂NRC(O)CH═CH—, —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-;        wherein R is H or optionally substituted C₁₋₆ aliphatic; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (g) L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,        —NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—,        —NHSO₂CH═CHCH₂—, —NHC(O)(C═N₂)—, —NHC(O)(C═N₂)C(O)—,        —NHC(O)CH═CHCH₂N(CH₃)—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,        —NHC(O)CH═CHCH₂O—, —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—,        —CH₂NHC(O)CH═CH—, —CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-;        and Y is hydrogen or C₁₋₆ aliphatic optionally substituted with        oxo, halogen, NO₂, or CN; or    -   (h) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one alkylidenyl double bond and at least        one methylene unit of L is replaced by —C(O)—, —NRC(O)—,        —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or        —C(O)O—, and one or two additional methylene units of L are        optionally and independently replaced by cyclopropylene, —O—,        —N(R)—, or —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (i) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one triple bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, or —C(O)O—, and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (j) L is —C≡C—, —C≡CCH₂N(isopropyl)-, —NHC(O)C≡CCH₂CH₂—,        —CH₂—C≡C≡CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or        —CH₂OC(═O)C≡C—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (k) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein one methylene unit of L is replaced by cyclopropylene        and one or two additional methylene units of L are independently        replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,        —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—; and Y is hydrogen or C₁₋₆        aliphatic optionally substituted with oxo, halogen, NO₂, or CN;        or    -   (l) L is a covalent bond and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (m) L is —C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (n) L is —N(R)C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   wherein each R, Q, Z, and R^(e) is as defined above and        described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (o) L is a bivalent C₁₋₈ saturated or unsaturated, straight or        branched, hydrocarbon chain; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (p) L is a covalent bond, —CH₂—, —NH—, —C(O)—, —CH₂NH—, —NHCH₂—,        —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,        —NHC(O)CH₂OC(O)—, or —SO₂NH—; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein.

In certain embodiments, the Y group of formula Ia or Ib is selected fromthose set forth in Table 3, below, wherein each wavy line indicates thepoint of attachment to the rest of the molecule.

TABLE 3 Exemplary Y groups

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

bb

cc

dd

ee

ff

gg

hh

ii

jj

kk

ll

mm

nn

oo

pp

qq

rr

ss

tt

uu

vv

ww

xx

yy

zz

aaa

bbb

ccc

ddd

eee

fff

ggg

hhh

iii

jjj

kkk

lll

mmm

nnn

ooo

ppp

qqq

rrr

sss

ttt

uuu

vvv

qqq

www

xxx

yyy

zzz

aaaa

bbbb

cccc

dddd

eeee

ffff

gggg

hhhh

iiii

jjjj

kkkk

llll

mmmm

nnnn

oooo

pppp

qqqq

rrrr

ssss

tttt

uuuu

vvvv

wwww

xxxx

yyyy

zzzz

aaaaa

bbbbb

ccccc

dddddwherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

In certain embodiments, R¹ is —C≡CH, —C≡CCH₂NH(isopropyl),—NHC(O)C≡CCH₂CH₃, —CH₂—C≡C≡CH₃, —C≡CCH₂OH, —CH₂C(O)C≡CH, —C(O)C≡CH, or—CH₂OC(═O)C≡CH. In some embodiments, R¹ is selected from —NHC(O)CH═CH₂,—NHC(O)CH═CHCH₂N(CH₃)₂, or —CH₂NHC(O)CH═CH₂.

In certain embodiments, R¹ is selected from those set forth in Table 4,below, wherein each wavy line indicates the point of attachment to therest of the molecule.

TABLE 4 Exemplary R¹ Groups

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

bb

cc

dd

ee

ff

gg

hh

ii

jj

kk

ll

mm

nn

oo

pp

qq

rr

ss

tt

uu

vv

ww

xx

yy

zz

aaa

bbb

ccc

ddd

eee

fff

ggg

hhh

iii

jjj

kkk

lll

mmm

nnn

ooo

ppp

qqq

rrr

sss

ttt

uuu

vvv

www

xxx

yyy

zzz

aaaa

bbbb

cccc

dddd

eeee

ffff

gggg

hhhh

iiii

jjjj

kkkk

llll

mmmm

nnnn

oooo

pppp

qqqq

rrrr

ssss

tttt

uuuu

vvvv

wwww

xxxx

yyyy

zzzz

aaaaa

bbbbb

ccccc

ddddd

eeeee

fffff

ggggg

hhhhh

iiiii

jjjjj

kkkkk

lllll

mmmmm

nnnnn

ooooo

ppppp

qqqqq

rrrrr

sssss

ttttt

uuuuu

vvvvv

wwwww

xxxxx

yyyyy

zzzzz

aaaaaa

bbbbbb

cccccc

dddddd

eeeeee

ffffff

gggggg

hhhhhh

iiiiii

jjjjjj

kkkkkk

llllll

mmmmmm

nnnnnn

oooooo

pppppp

qqqqqq

rrrrrr

ssssss

tttttt

uuuuuu

vvvvvv

wwwwww

xxxxxx

yyyyyywherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

As defined generally above, R¹ is a warhead group, or, when R¹ and R^(x)form a ring, then -Q-Z is a warhead group. Without wishing to be boundby any particular theory, it is believed that such R¹ groups, i.e.warhead groups, are particularly suitable for covalently binding to akey cysteine residue in the binding domain of certain protein kinases.Protein kinases having a cysteine residue in the binding domain areknown to one of ordinary skill in the art and include ErbB1, ErbB2, andErbB4, or a mutant thereof. In certain embodiments, compounds of thepresent invention have a warhead group characterized in that inventivecompounds target one or more of the following cysteine residues:

ERBB1 ITQLMPFG C LLDYVREH ERBB2 VTQLMPYG C LLDHVREN ERBB4 VTQLMPHG CLLEYVHEH

Thus, in some embodiments, R¹ is characterized in that the -L-Y moietyis capable of covalently binding to a cysteine residue therebyirreversibly inhibiting the enzyme. In certain embodiments, the cysteineresidue is Cys797 of ErbB1, Cys805 of ErbB2 and Cys803 of ErbB4, or amutant thereof, where the provided residue numbering is in accordancewith Uniprot (code POO533 for ErbB1; code PO4626 for ErbB2, and Q15303for ErbB4). It will be understood that the Cys of ErbB1 (EGFR) isvariably called 773 or 797 depending on whether the parent sequencecontains the signal peptide or not. Thus, in accordance with the presentinvention, the relevant cysteine residue of ErbB1 may be described asCys 773 or Cys 797 and these terms are used interchangeably.

One of ordinary skill in the art will recognize that a variety ofwarhead groups, as defined herein, are suitable for such covalentbonding. Such R¹ groups include, but are not limited to, those describedherein and depicted in Table 4, infra.

As depicted in formulae I-a and I-b supra, the R¹ warhead group can bein an ortho-, meta-, or para-position. In certain embodiments, the R¹warhead group is in a meta-position of the phenyl ring relative to therest of the molecule.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of TEC, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 449.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of BTK, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 481.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of ITK, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 442.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of BMX, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 496.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of JAK3, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 909.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of TXK, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 350.

One of ordinary skill in the art will recognize that a variety ofwarhead groups, as defined herein, are suitable for such covalentbonding. Such R¹ groups include, but are not limited to, those describedherein and depicted in Table 3, infra.

Exemplary compounds of the present invention are set forth in Table 5below.

TABLE 5 Exemplary Compounds

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

I-32

I-33

I-34

I-35

I-36

I-37

I-38

I-39

I-40

I-41

I-42

I-43

I-44

I-45

I-46

I-47

I-48

I-49

I-50

I-51

I-52

I-53

I-54

I-55

I-56

I-57

I-58

I-59

I-60

I-61

I-62

I-63

I-64

I-65

I-66

I-67

I-68

I-69

I-70

I-71

I-72

I-73

I-74

I-75

I-76

I-77

I-78

I-79

I-80

I-81

I-82

I-83

I-84

I-85

I-86

I-87

I-88

I-89

I-90

I-91

I-92

I-93

I-94

I-95

I-96

I-97

I-98

I-99

I-100

I-101

I-102

I-103

I-104

I-105

I-106

I-107

I-108

I-109

I-110

I-111

I-112

I-113

I-114

I-115

I-116

I-117

I-118

I-119

I-120

I-121

I-122

I-123

I-124

I-125

I-126

I-127

I-128

I-129

I-130

I-131

I-132

I-133

I-134

I-135

I-136

I-137

I-138

I-139

I-140

I-141

I-142

I-143

I-144

I-145

I-146

I-147

I-148

I-149

I-150

I-151

I-152

I-153

I-154

I-155

I-156

I-157

I-158

I-159

I-160

I-161

I-162

I-163

I-164

I-165

I-166

I-167

I-168

I-169

I-170

I-171

I-172

I-173

I-174

I-175

I-176

I-177

I-178

I-179

I-180

I-181

I-182

I-183

I-184

I-185

I-186

I-187

I-188

I-189

I-190

I-191

I-192

I-193

I-194

I-195

I-196

I-197

I-198

I-199

I-200

I-201

I-202

I-203

I-204

I-205

I-206

I-207

I-208

I-209

I-210

I-211

I-212

I-213

I-214

I-215

I-216

I-217

I-218

I-219

I-220

I-221

I-222

I-223

I-224

I-225

I-226

I-227

I-228

I-229

I-230

I-231

I-232

I-233

I-234

I-235

I-236

I-237

I-238

I-239

I-240

I-241

I-242

I-243

I-244

I-245

I-246

I-247

I-248

I-249

I-250

I-251

I-252

I-253

I-254

I-255

I-256

I-257

I-258

I-259

I-260

I-261

I-262

I-263

I-264

I-265

I-266

I-267

I-268

I-269

I-270

I-271

I-272

I-273

I-274

I-275

I-276

I-277

I-278

I-279

I-280

I-281

I-282

I-283

I-284

I-285

I-286

I-287

I-288

I-289

I-290

I-291

I-292

I-293

I-294

I-295

I-296

I-297

I-298

I-299

I-300

I-301

I-302

I-303

I-304

I-305

I-306

I-307

I-308

I-309

I-310

I-311

I-312

I-313

I-314

I-315

I-316

I-317

I-318

I-319

I-320

I-321

I-322

I-323

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I-325

I-326

I-327

I-328

I-329

I-330

I-331

I-332

I-333

I-334

I-335

I-336

I-337

I-338

I-339

I-340

I-341

I-342

I-343

I-344

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I-346

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I-348

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I-350

I-351

I-352

I-353

I-354

I-355

I-356

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I-358

I-359

I-360

I-361

I-362

I-363

I-364

I-365

I-366

I-367

I-368

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I-371

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I-377

I-378

I-379

I-380

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I-386

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I-388

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I-393

In certain embodiments, the present invention provides any compounddepicted in Table 5, above, or a pharmaceutically acceptable saltthereof.

In certain embodiments, the present invention provides a compoundselected from:

or a pharmaceutically acceptable salt thereof.

As described herein, compounds of the present invention are irreversibleinhibitors of at least one of ErbB1, ErbB2, ErbB3 and ErbB4, or a mutantthereof. In some embodiments, provided compounds are irreversibleinhibitors of a TEC-kinase (e.g. BTK) and JAK3. One of ordinary skill inthe art will recognize that certain compounds of the present inventionare reversible inhibitors. In certain embodiments, such compounds areuseful as assay comparator compounds. In other embodiments, suchreversible compounds are useful as inhibitors of ErbB1, ErbB2, ErbB3,ErbB4, a TEC-kinase, and/or JAK3, or a mutant thereof, and thereforeuseful for treating one or disorders as described herein. An exemplaryreversible compound of the present invention has the followingstructure.

or a pharmaceutically acceptable salt thereof.

4. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in compositions of this invention issuch that is effective to measurably inhibit a protein kinase,particularly at least one of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase,and/or JAK3, or a mutant thereof, in a biological sample or in apatient. In certain embodiments, the amount of compound in compositionsof this invention is such that is effective to measurably inhibit atleast one of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or amutant thereof, in a biological sample or in a patient. In certainembodiments, a composition of this invention is formulated foradministration to a patient in need of such composition. In someembodiments, a composition of this invention is formulated for oraladministration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof at least one of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/orJAK3, or a mutant thereof.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. Preferably, provided compositions should be formulatedso that a dosage of between 0.01-100 mg/kg body weight/day of theinhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for theinhibition of protein kinase activity of one or more enzymes.

Drug resistance is emerging as a significant challenge for targetedtherapies. For example, drug resistance has been reported for Gleevec®and Iressa®, as well as several other kinase inhibitors in development.In addition, drug resistance has been reported for the cKit and PDGFRreceptors. It has been reported that irreversible inhibitors may beeffective against drug resistant forms of protein kinases (Kwak, E. L.,R. Sordella, et al. (2005). “Irreversible inhibitors of the EGF receptormay circumvent acquired resistance to gefitinib.” PNAS 102(21):7665-7670.) Without wishing to be bound by any particular theory, it isbelieved that compounds of the present invention may be effectiveinhibitors of drug resistant forms of protein kinases.

As used herein, the term “clinical drug resistance” refers to the lossof susceptibility of a drug target to drug treatment as a consequence ofmutations in the drug target.

As used herein, the term “resistance” refers to changes in the wild-typenucleic acid sequence coding a target protein, and/or the proteinsequence of the target, which changes decrease or abolish the inhibitoryeffect of the inhibitor on the target protein.

Examples of kinases that are inhibited by the compounds and compositionsdescribed herein and against which the methods described herein areuseful include ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase (including BTK,ITK, TEC, BMX and RLK), and/or JAK3, or a mutant thereof.

The activity of a compound utilized in this invention as an inhibitor ofErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or a mutantthereof, may be assayed in vitro, in vivo or in a cell line. In vitroassays include assays that determine inhibition of either thephosphorylation activity and/or the subsequent functional consequences,or ATPase activity of activated ErbB1, ErbB2, ErbB3, ErbB4, aTEC-kinase, and/or JAK3, or a mutant thereof. Alternate in vitro assaysquantitate the ability of the inhibitor to bind to ErbB1, ErbB2, ErbB3,ErbB4, a TEC-kinase, and/or JAK3. Inhibitor binding may be measured byradiolabeling the inhibitor prior to binding, isolating theinhibitor/ErbB1, inhibitor/ErbB2, inhibitor/ErbB3, inhibitor/ErbB4,inhibitor/TEC-kinase (i.e., TEC, BTK, ITK, RLK and BMX), orinhibitor/JAK3 complex and determining the amount of radiolabel bound.Alternatively, inhibitor binding may be determined by running acompetition experiment where new inhibitors are incubated with ErbB1,ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3 bound to knownradioligands. Detailed conditions for assaying a compound utilized inthis invention as an inhibitor of ErbB1, ErbB2, ErbB3, ErbB4, aTEC-kinase, and/or JAK3, or a mutant thereof, are set forth in theExamples below.

Protein tyrosine kinases are a class of enzymes that catalyze thetransfer of a phosphate group from ATP or GTP to a tyrosine residuelocated on a protein substrate. Receptor tyrosine kinases act totransmit signals from the outside of a cell to the inside by activatingsecondary messaging effectors via a phosphorylation event. A variety ofcellular processes are promoted by these signals, includingproliferation, carbohydrate utilization, protein synthesis,angiogenesis, cell growth, and cell survival.

(a) ErbB Family

ErbB receptors, a major family of receptor tyrosine kinases, arecomposed of an extracellular ligand binding domain, a singletransmembrane domain, and an intracellular domain with tyrosine kinaseactivity. The ErbB family comprises ErbB1 (commonly known as EGFR),ErbB2 (commonly known as HER2 or neu), ErbB3 (commonly known as HER3),and ErbB4 (commonly known as HER4). More than 10 ligands (including EGF,TGFα, AR, BTC, EPR, HB-EGF, NRG-1, NRG-2, NRG-3, NRG-4) have beenidentified for the various receptor family members. Upon ligand bindingthe extracellular domain undergoes conformational change, allowing theformation of homodimers or heterodimers with other members of the ErbBfamily. Dimerization induces tyrosine phosphorylation of specificresidues in the intracellular domain that serve as docking sites foradaptor proteins and downstream effectors. In some contexts, activationof phosphatidyl-inositol 3-kinase (PI3K) and mitogen-activated proteinkinase pathways occur, leading to cell proliferation and survival (Lin,N. U.; Winer, E. P., Breast Cancer Res 6: 204-210, 2004).

Interaction between family members is necessitated by deficiencies inErbB2, which has no known ligand, and ErbB3, which is kinase dead. EGFR,ErbB3, and ErbB4 bind ligand to induce ErbB receptor homodimerization orheterodimerization, whereas ErbB2 functions as the preferreddimerization partner. The composition of the pairwise combinations isimportant for signal diversification, as dimer identity determines whichdownstream pathways are activated. Representative downstream geneproducts in the ErbB signal transduction pathway include Shc, Grb2,SOS1, Ras, Raf1, Mek, ERK1, ERK2, ERa, Akt, mTOR, FKHR, p27, Cyclin D1,FasL, GSK-3, Bad, and STAT3.

There is strong precedent for involvement of the EGFR and other membersof the ErbB family in human cancer because over 60% of all solid tumorsoverexpress at least one of these proteins or their ligands.Constitutively active, tumorigenic EGFR vIII, a mutant possessing atruncated extracellular domain, has been reported to be present in up to78% of breast carcinomas and has also been found in glioblastomas.Overexpression of EGFR is commonly found in breast, lung, head and neck,bladder tumors, while ErbB2 expression is frequently elevated in humantumors of epithelial origin. Activating mutations in the tyrosine kinasedomain have been identified in patients with non-small cell lung cancer(Lin, N. U.; Winer, E. P., Breast Cancer Res 6: 204-210, 2004). ErbB1and/or ErbB2 amplification has also been implicated in squamous cellcarcinomas, salivary gland carcinomas, ovarian carcinomas, andpancreatic cancers (Cooper, G. C. Oncogenes. 2^(nd) ed. Sudbury: Jonesand Barlett, 1995; Zhang, Y., et al., Cancer Res 66: 1025-32, 2006).Overexpression of ErbB2 has potent transforming activity, likely due toits ability to cooperate with other ErbB receptors (Sherman, L., et al.,Oncogene 18: 6692-99, 1999). In fact, some human cancers thatoverexpress both EGFR and ErbB2 have a poorer prognosis than cancersthat overexpress either receptor alone.

The ErbB signaling network is often a key component in the pathogenesisof breast cancer. Amplification of ErbB2 is associated with anaggressive tumor phenotype that is characterized by relatively rapidtumor growth, metastatic spread to visceral sites, and drug resistance.ErbB2 has been shown to be amplified in 20% of axillary node-negative(“ANN”) breast cancer cases, and this amplification has been identifiedas an independent prognostic factor for risk of recurrence in ANN breastcancer. (Andrulis, I. L., et al., J Clin Oncol 16: 1340-9, 1998).

Targeted blockade of ErbB signaling with trastuzumab (Herceptin), amonoclonal antibody directed at ErbB2, has been shown to improvesurvival in women with ErbB2-positive, advanced breast cancer. Othermonoclonal antibodies directed against ErbB receptors include cetuximab(Erbitux) and panitumumab (Vectibix).

Several small molecule tyrosine kinase inhibitors (TKIs) have been foundto act selectively upon ErbB family members. Notable examples includegefitinib (Iressa) and erlotinib (Tarceva), both of which target theEGFR. These small molecules compete with ATP for binding to the kinasedomain of the receptor. Compared to monoclonal antibodies, TKIs haveseveral advantages in that they are orally bioavailable, well-tolerated,and appear to be active against truncated forms of ErbB2 and EGFRreceptors (e.g., EGFR vIII) in vitro. In addition, the small size ofsmall molecule TKIs may allow them to penetrate sanctuary sites such asthe central nervous system. Finally, the homology between kinase domainsof ErbB receptors allows for development of TKIs that target more thanone member of the ErbB family simultaneously, the advantages of whichare described herein.

Although certain malignancies have been linked to the overexpression ofindividual receptors, efficient signal transduction relies on thecoexpression of ErbB receptor family members. This cooperation of ErbBreceptor family members in signal transduction and malignanttransformation may limit the success of agents that target individualreceptors in the treatment of cancer; a potential mechanism ofresistance to agents targeting a single ErbB receptor is upregulation ofother members of the receptor family (Britten, C. D., Mol Cancer Ther 3:1335-42, 2004).

Agents that target two or more ErbB receptors are called pan-ErbBregulators. ERRP is a pan-ErbB negative regulator that is expressed inmost benign pancreatic ductal epithelium and islet cells. Tumors havebeen found to experience a progressive loss in ERRP expression. ThatErbitux and Herceptin show success in a limited patient base (tumorshaving increased expression of EGFR or ErbB2) could be partly due tocoexpression of multiple ErbB family members.

In both in vitro and in vivo models, strategies that employ a dual ErbBapproach seem to have greater antitumor activity than agents targeting asingle ErbB receptor. Thus, agents that target multiple members of ErbBfamily are likely to provide therapeutic benefit to a broader patientpopulation (Zhang, Y., et al., Cancer Res 66: 1025-32, 2006). In certainembodiments, provided compounds inhibit one or more of ErbB1, ErbB2,ErbB3, and ErbB4. In some embodiments, provided compounds inhibit two ormore of ErbB1, ErbB2, ErbB3, and ErbB4, or a mutant thereof, and aretherefore pan-ErbB inhibitors.

Clearly, there is growing evidence to support the concurrent inhibitionof two or more ErbB (i.e., pan-ErbB) receptors in cancer therapy.Possible pan-ErbB approaches with small molecules include usingcombinations of agents that target individual ErbB receptors, usingsingle agents that target multiple ErbB receptors, or using agents thatinterfere with ErbB receptor interactions (e.g., dimerization).Additional strategies include therapies utilizing a small molecule incombination with antibodies, or chemoprevention therapies (Lin, N. U.;Winer, E. P., Breast Cancer Res 6: 204-210, 2004).

An example of small molecule pan-ErbB inhibition is CI-1033, anirreversible pan-ErbB inhibitor that covalently binds to the ATP bindingsite of the intracellular kinase domain. Another irreversible pan-ErbBreceptor tyrosine kinase inhibitor is HKI-272, which inhibits the growthof tumor cells that express ErbB-1 (EGFR) and ErbB-2 (HER-2) in cultureand xenografts, and has antitumor activity in HER-2-positive breastcancer (Andrulis, I. L., et al., J Clin Oncol 16: 1340-9, 1998).Irreversible inhibitors have demonstrated superior antitumor activity incomparison with reversible inhibitors.

Neurofibromatosis type I (NF1) is a dominantly inherited human diseaseaffecting one in 2500-3500 individuals. Several organ systems areaffected, including bones, skin, iris, and the central nervous system,as manifested in learning disabilities and gliomas. A hallmark of NF1 isthe development of benign tumors of the peripheral nervous system(neurofibromas), which vary greatly in both number and size amongpatients. Neurofibromas are heterogeneous tumors composed of Schwanncells, neurons, fibroblasts and other cells, w/Schwann cells being themajor (60-80%) cell type.

Abberant expression of the EGFR is associated with tumor development inNF1 and in animal models of NF1, suggesting a role in pathogenesis andrepresenting a novel potential therapeutic target. EGFR expressionaffects the growth of tumor cell lines derived from NF1 patients underconditions where EGF is not the primary factor driving growth of thecells. These data suggest that EGFR may play an important role in NF1tumorigenesis and Schwann cell transformation (DeClue, J. E., et al., JClin Invest 105: 1233-41, 2000).

Patients with NF1 develop aggressive Schwann cell neoplasmas known asmalignant peripheral nerve sheath tumors (MPNSTs). Schwann cells are themajor supportive cell population in the peripheral nervous system.Neoplastic Schwann cells within these neoplasms variably express theErbB tyrosine kinases mediating NRG-1 responses (ErbB2, ErbB3, ErbB4).Neuregulin-1 (NRG-1) proteins promote the differentiation, survival,and/or proliferation of many cell types in the developing nervoussystem, and overexpression of NRG-1 in myelinating Schwann cells inducesthe formation of malignant peripheral nerve sheath tumors (MPNSTs)(Fallon, K. B., et al., J Neuro Oncol 66: 273-84, 2004).

Deregulation of Schwann cell growth is a primary defect driving thedevelopment of both benign neurofibromas and MPNST in neurofibromatosistype I (NF1) patients. Growth of MPNSTs and transformed mouse Schwanncells in vitro is highly EGF-dependent and can be blocked by EGFRinhibitors under conditions where EGF is the primary growth factor. Somehuman MPNST cell lines have been found to demonstrate constitutive ErbBphosphorylation. While treatment with ErbB inhibitors abolishes ErbBphosphorylation and reduces DNA synthesis in these lines, effectivechemotherapeutic regimens for MPNST remain elusive (Stonecypher, M. S.,et al., Oncogene 24: 5589-5605, 2005).

Schwannomas are peripheral nerve tumors comprised almost entirely ofSchwann-like cells, and typically have mutations in theneurofibromatosis type II (NF2) tumor suppressor gene. Ninety percent ofNF2 patients develop bilateral vestibular schwannomas and/or spinalschwannomas. Enlarging schwannomas can compress adjacent structures,resulting in deafness and other neurologic problems. Surgical removal ofthese tumors is difficult, often resulting in increased patientmorbidity.

Both normal human Schwann cells and schwannoma cells express neuregulinreceptors (i.e., ErbB receptors), and schwannoma cells proliferate inresponse to neuregulin. It is possible that aberrant neuregulinproduction or response contributes to aberrant schwannoma cellproliferation (Pelton, P. D., et al., Oncogene 17: 2195-2209, 1998).

The NF2 tumor suppressor, Merlin, is a membrane/cytoskeleton-associatedprotein implicated in the regulation of tyrosine kinase activity.Genetic interactions between a Merlin mutation and EGFR pathwaymutations have been documented in Drosophila (LaJeunesse, D. R., et al.,Genetics 158: 667-79, 2001). Other evidence suggests Merlin can inhibitEGFR internalization and signaling upon cell-cell contact by restrainingthe EGFR into a membrane compartment from which it can neither signalnor be internalized (McClatchey, A. I., et al., Genes and Development19: 2265-77, 2005; Curto, M. C., et al., J Cell Biol 177: 893-903,2007).

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

Provided compounds are inhibitors of one or more of ErbB1, ErbB2, ErbB3,and ErbB4 and are therefore useful for treating one or more disordersassociated with activity of one of more of ErbB1, ErbB2, ErbB3, andErbB4. Thus, in certain embodiments, the present invention provides amethod for treating an ErbB1-mediated, an ErbB2-mediated, anErbB3-mediated, and/or ErbB4-mediated disorder comprising the step ofadministering to a patient in need thereof a compound of the presentinvention, or pharmaceutically acceptable composition thereof.

As used herein, the terms “ErbB1-mediated”, “ErbB2-mediated,”“ErbB3-mediated,” and/or “ErbB4-mediated” disorders or conditions asused herein means any disease or other deleterious condition in whichone or more of ErbB1, ErbB2, ErbB3, and/or ErbB4, or a mutant thereof,are known to play a role. Accordingly, another embodiment of the presentinvention relates to treating or lessening the severity of one or morediseases in which one or more of ErbB1, ErbB2, ErbB3, and/or ErbB4, or amutant thereof, are known to play a role. Specifically, the presentinvention relates to a method of treating or lessening the severity of adisease or condition selected from a proliferative disorder, whereinsaid method comprises administering to a patient in need thereof acompound or composition according to the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more disorders selectedfrom a cancer. In some embodiments, the cancer is associated with asolid tumor. In certain embodiments, the cancer is breast cancer,glioblastoma, lung cancer, cancer of the head and neck, colorectalcancer, bladder cancer, or non-small cell lung cancer. In someembodiments, the present invention provides a method for treating orlessening the severity of one or more disorders selected from squamouscell carcinoma, salivary gland carcinoma, ovarian carcinoma, orpancreatic cancer.

In certain embodiments, the present invention provides a method fortreating or lessening the severity of neurofibromatosis type I (NF1),neurofibromatosis type II (NF2) Schwann cell neoplasms (e.g. MPNST's),or Schwannomas.

(b) TEC Family

The TEC family of non-receptor tyrosine kinases, referred to herein as“TEC-kinases,” plays a central role in signaling throughantigen-receptors such as the TCR, BCR and Fc receptors (reviewed inMiller A, et al. Current Opinion in Immunology 14; 331-340 (2002).TEC-kinases are essential for T cell activation. Three members of thefamily, Itk, Rlk and, are activated downstream of antigen receptorengagement in T cells and transmit signals to downstream effectors,including PLC-γ. Combined deletion of Itk and Rlk in mice leads to aprofound inhibition of TCR responses including proliferation, cytokineproduction and immune responses to an intracellular parasite (Toxoplasmagondii) (Schaeffer et al., Science 284; 638-641 (1999)). Intracellularsignalling following TCR engagement is effected in ITK/RLK deficient Tcells; inositol triphosphate production, calcium mobilization and MAPkinase activation are all reduced. Tec-kinases are also essential for Bcell development and activation.

TEC-kinases include five family members, which are expressed primarilyin hematopoietic cells: TEC, BTK, ITK (also known as TSK and EMT), RLK(also known as TXK), and BMX (also known as ETK). Additional relatedTEC-kinases have been found in Drosophila melanogaster, zebrafish (Daniorerió), skate (Raja eglanteria), and sea urchin (Anthocidariscrassispina).

Provided compounds are inhibitors of one of more TEC-kinases and aretherefore useful for treating one or more disorders associated withactivity of one or more TEC-kinases. Thus, in certain embodiments, thepresent invention provides a method for treating a TEC-mediated disordercomprising the step of administering to a patient in need thereof acompound of the present invention, or pharmaceutically acceptablecomposition thereof.

The term “TEC-mediated condition”, as used herein means any disease orother deleterious condition in which TEC-kinases are known to play arole. Such conditions include those described herein and in Melcher, Met al., “The Role of TEC Family Kinases in Inflammatory Processes”,Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, Vol. 6,No. 1, pp. 61-69 (February 2007). Accordingly, another embodiment of thepresent invention relates to treating or lessening the severity of oneor more diseases in which TEC-kinases are known to play a role.Specifically, the present invention relates to a method of treating orlessening the severity of a disease or condition selected fromautoimmune, inflammatory, proliferative, and hyperproliferative diseasesand immunologically-mediated diseases including rejection oftransplanted organs or tissues and Acquired Immunodeficiency Syndrome(AIDS)(also known as HIV), wherein said method comprises administeringto a patient in need thereof a composition of the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases of therespiratory tract including, without limitation, reversible obstructiveairways diseases including asthma, such as bronchial, allergic,intrinsic, extrinsic and dust asthma, particularly chronic or inveterateasthma (e.g., late asthma airways hyper-responsiveness) and bronchitis.In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including those conditionscharacterized by inflammation of the nasal mucus membrane, includingacute rhinitis, allergic, atrophic rhinitis and chronic rhinitisincluding rhinitis caseosa, hypertrophic rhinitis, rhinitis purulenta,rhinitis sicca and rhinitis medicamentosa; membranous rhinitis includingcroupous, fibrinous and pseudomembranous rhinitis and scrofoulousrhinitis, seasonal rhinitis including rhinitis nervosa (hay fever) andvasomotor rhinitis, sarcoidosis, farmer's lung and related diseases,fibroid lung, and idiopathic interstitial pneumonia.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases of the boneand joints including, without limitation, rheumatoid arthritis,seronegative spondyloarthropathies (including ankylosing spondylitis,psoriatic arthritis and Reiter's disease), Behcet's disease, Sjogren'ssyndrome, systemic sclerosis, osteoporosis, bone cancer, and bonemetastasis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases and disordersof the skin, including, without limitation, psoriasis, systemicsclerosis, atopical dermatitis, contact dermatitis and other eczematousdermatitis, seborrhoetic dermatitis, Lichen planus, pemphigus, bullouspemphigus, epidermolysis bullosa, urticaria, angiodermas, vasculitides,erythemas, cutaneous eosinophilias, uveitis, alopecia, areata and vernalconjunctivitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases and disordersof the gastrointestinal tract, including, without limitation, celiacdisease, proctitis, eosinophilic gastro-enteritis, mastocytosis,pancreatitis, Crohn's disease, ulcerative colitis, food-relatedallergies which have effects remote from the gut, e. g. migraine,rhinitis and eczema.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including those diseases anddisorders of other tissues and systemic disease, including, withoutlimitation, multiple sclerosis, artherosclerosis, lupus erythematosus,systemic lupus erythematosus, Hashimoto's thyroiditis, myastheniagravis, diabetes, nephrotic syndrome, eosinophilia fascitis, hyper IgEsyndrome, lepromatous leprosy, sezary syndrome and idiopathicthrombocytopenia purpura, restenosis following angioplasty, tumours (forexample leukemia, lymphomas, and prostate cancers), andartherosclerosis. In certain embodiments, the diabetes is type Idiabetes.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including allograft rejectionincluding, without limitation, acute and chronic allograft rejectionfollowing for example transplantation of kidney, heart, liver, lung,bone marrow, skin and cornea; and chronic graft versus host disease.

In some embodiments, the present invention relates to a method oftreating or lessening the severity of one or more of the diseases orconditions associated with TEC-kinases, as recited above, wherein saidmethod comprises administering to a patient in need thereof a compoundor composition according to the present invention.

(c) Bruton's Tvrosine Kinase (BTK)

Bruton's tyrosine kinase (“BTK”), a member of TEC-kinases, is a keysignaling enzyme expressed in all hematopoietic cell types except Tlymphocytes and natural killer cells. BTK plays an essential role in theB-cell signaling pathway linking cell surface B-cell receptor (BCR)stimulation to downstream intracellular responses.

BTK is a key regulator of B-cell development, activation, signaling, andsurvival (Kurosaki, Curr Op Imm, 2000, 276-281; Schaeffer andSchwartzberg, Curr Op Imm 2000, 282-288). In addition, BTK plays a rolein a number of other hematopoietic cell signaling pathways, e.g., Tolllike receptor (TLR) and cytokine receptor-mediated TNF-α production inmacrophages, IgE receptor (Fc_epsilon_RI) signaling in mast cells,inhibition of Fas/APO-1 apoptotic signaling in B-lineage lymphoid cells,and collagen-stimulated platelet aggregation. See, e.g., C. A. Jeffries,et al., (2003), Journal of Biological Chemistry 278:26258-26264; N. J.Horwood, et al., (2003), The Journal of Experimental Medicine 197:1603-1611; Iwaki et al. (2005), Journal of Biological Chemistry280(48):40261-40270; Vassilev et al. (1999), Journal of BiologicalChemistry 274(3): 1646-1656, and Quek et al. (1998), Current Biology8(20): 1137-1140.

Patients with mutations in BTK have a profound block in B celldevelopment, resulting in the almost complete absence of mature Blymphocytes and plasma cells, severely reduced Ig levels and a profoundinhibition of humoral response to recall antigens (reviewed in Vihinenet al Frontiers in Bioscience 5: d917-928). Mice deficient in BTK alsohave a reduced number of peripheral B cells and greatly decreased serumlevels of IgM and IgG3. BTK deletion in mice has a profound effect on Bcell proliferation induced by anti-IgM, and inhibits immune responses tothymus-independent type II antigens (Ellmeier et al, J Exp Med 192:1611-1623 (2000)). BTK also plays a crucial role in mast cell activationthrough the high-affinity IgE receptor (Fc_epsilon_RI). BTK deficientmurine mast cells have reduced degranulation and decreased production ofproinflammatory cytokines following Fc_epsilon_RI cross-linking(Kawakami et al. Journal of Leukocyte Biology 65: 286-290).

BTK has been implicated in diabetes. BTK deficiency in non-obesediabetic mice dramatically protects against diabetes and improves Bcell-related tolerance, as indicated by failure to generateautoantibodies to insulin (Kendall, et al. J. Immunol. 183: 6403-6412(2009)). Modulation of BTK and improvement of B cell-related tolerancecan therefore be used in treatment of diabetes, particularly Tcell-mediated autoimmune diabetes, e.g. type I diabetes.

BTK is also implicated in various cancers. For example, BTK isupregulated in pancreatic cancer cells compared with normal pancreascells, and BTK is also upregulated in chronic pancreatitis cells, whichis sometimes a precursor to pancreatic cancer (Crnogorac-Jurcevic, etal. Gastroenterology 129: 1454-1463 (2005)). Due to the key role of BTKin regulation of B-cell development, activation, signaling, andsurvival, BTK is involved in many B cell-related cancers.

Provided compounds are inhibitors of BTK and are therefore useful fortreating one or more disorders associated with activity of BTK. Thus, insome embodiments, the present invention provides a method for treating aBTK-mediated disorder comprising the step of administering to a patientin need thereof a compound of the present invention, or pharmaceuticallyacceptable composition thereof.

As used herein, the term “BTK-mediated” disorders or conditions as usedherein means any disease or other deleterious condition in which BTK, ora mutant thereof, is known to play a role. Accordingly, anotherembodiment of the present invention relates to treating or lessening theseverity of one or more diseases in which BTK, or a mutant thereof, isknown to play a role. Specifically, the present invention relates to amethod of treating or lessening the severity of a disease or conditionselected from a proliferative disorder or an autoimmune disorder,wherein said method comprises administering to a patient in need thereofa compound or composition according to the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK. In some embodiments, the disease orcondition is an autoimmune disease, e.g., inflammatory bowel disease,arthritis, systemic lupus erythematosus (SLE), vasculitis, idiopathicthrombocytopenic purpura (ITP), rheumatoid arthritis, psoriaticarthritis, osteoarthritis, Still's disease, juvenile arthritis,diabetes, myasthenia gravis, Hashimoto's thyroiditis, Ord's thyroiditis,Graves' disease, autoimmune thyroiditis, Sjogren's syndrome, multiplesclerosis, systemic sclerosis, Lyme neuroborreliosis, Guillain-Barresyndrome, acute disseminated encephalomyelitis, Addison's disease,opsoclonus-myoclonus syndrome, ankylosing spondylosis, antiphospholipidantibody syndrome, aplastic anemia, autoimmune hepatitis, autoimmunegastritis, pernicious anemia, celiac disease, Goodpasture's syndrome,idiopathic thrombocytopenic purpura, optic neuritis, scleroderma,primary biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis,temporal arteritis, warm autoimmune hemolytic anemia, Wegener'sgranulomatosis, psoriasis, alopecia universalis, Behcet's disease,chronic fatigue, dysautonomia, membranous glomerulonephropathy,endometriosis, interstitial cystitis, pemphigus vulgaris, bullouspemphigoid, neuromyotonia, scleroderma, or vulvodynia. In someembodiments, the disease or condition is a hyperproliferative disease orimmunologically-mediated diseases including rejection of transplantedorgans or tissues and Acquired Immunodeficiency Syndrome (AIDS, alsoknown as HIV). In certain embodiments, the diabetes is type I diabetes.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from heteroimmune conditions or diseases, which include, butare not limited to graft versus host disease, transplantation,transfusion, anaphylaxis, allergies (e.g., allergies to plant pollens,latex, drugs, foods, insect poisons, animal hair, animal dander, dustmites, or cockroach calyx), type I hypersensitivity, allergicconjunctivitis, allergic rhinitis, and atopic dermatitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from an inflammatory disease, e.g., asthma, appendicitis,atopic dermatitis, asthma, allergy, blepharitis, bronchiolitis,bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, chronicgraft rejection, colitis, conjunctivitis, Crohn's disease, cystitis,dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis,endometritis, enteritis, enterocolitis, epicondylitis, epididymitis,fasciitis, fibrositis, gastritis, gastroenteritis, Henoch-Schonleinpurpura, hepatitis, hidradenitis suppurativa, immunoglobulin Anephropathy, interstitial lung disease, laryngitis, mastitis,meningitis, myelitis myocarditis, myositis, nephritis, oophoritis,orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis,peritonitis, pharyngitis, pleuritis, phlebitis, pneumonitis, pneumonia,polymyositis, proctitis, prostatitis, pyelonephritis, rhinitis,salpingitis, sinusitis, stomatitis, synovitis, tendonitis, tonsillitis,ulcerative colitis, uveitis, vaginitis, vasculitis, or vulvitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from a cancer. In one embodiment, the cancer is a B-cellproliferative disorder, e.g., diffuse large B cell lymphoma, follicularlymphoma, chronic lymphocytic lymphoma, chronic lymphocytic leukemia,acute lymphocytic leukemia, B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenicmarginal zone lymphoma, multiple myeloma (also known as plasma cellmyeloma), non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmacytoma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, mantle cell lymphoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, Burkitt lymphoma/leukemia, or lymphomatoid granulomatosis. Insome embodiments, the cancer is breast cancer, prostate cancer, orcancer of the mast cells (e.g., mastocytoma, mast cell leukemia, mastcell sarcoma, systemic mastocytosis). In one embodiment, the cancer isbone cancer. In another embodiment, the cancer is of other primaryorigin and metastasizes to the bone. In certain embodiments, the canceris colorectal cancer or pancreatic cancer.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases or conditionsassociated with BTK including diseases of the bone and joints including,without limitation, rheumatoid arthritis, seronegativespondyloarthropathies (including ankylosing spondylitis, psoriaticarthritis and Reiter's disease), Behcet's disease, Sjogren's syndrome,systemic sclerosis, osteoporosis, bone cancer, and bone metastasis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from a thromboembolic disorder, e.g., myocardial infarct,angina pectoris, reocclusion after angioplasty, restenosis afterangioplasty, reocclusion after aortocoronary bypass, restenosis afteraortocoronary bypass, stroke, transitory ischemia, a peripheral arterialocclusive disorder, pulmonary embolism, or deep venous thrombosis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, including infectious and noninfectiousinflammatory events and autoimmune and other inflammatory diseases.These autoimmune and inflammatory diseases, disorders, and syndromesinclude inflammatory pelvic disease, urethritis, skin sunburn,sinusitis, pneumonitis, encephalitis, meningitis, myocarditis,nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis,dermatitis, gingivitis, appendicitis, pancreatitis, cholocystitus,agammaglobulinemia, psoriasis, allergy, Crohn's disease, irritable bowelsyndrome, ulcerative colitis, Sjogren's disease, tissue graft rejection,hyperacute rejection of transplanted organs, asthma, allergic rhinitis,chronic obstructive pulmonary disease (COPD), autoimmune polyglandulardisease (also known as autoimmune polyglandular syndrome), autoimmunealopecia, pernicious anemia, glomerulonephritis, dermatomyositis,multiple sclerosis, scleroderma, vasculitis, autoimmune hemolytic andthrombocytopenic states, Goodpasture's syndrome, atherosclerosis,Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes,septic shock, systemic lupus erythematosus (SLE), rheumatoid arthritis,psoriatic arthritis, juvenile arthritis, osteoarthritis, chronicidiopathic thrombocytopenic purpura, Waldenstrom macroglobulinemia,myasthenia gravis, Hashimoto's thyroiditis, atopic dermatitis,degenerative joint disease, vitiligo, autoimmune hypopituitarism,Guillain-Barre syndrome, Behcet's disease, scleraderma, mycosisfungoides, acute inflammatory responses (such as acute respiratorydistress syndrome and ischemia/reperfusion injury), and Graves' disease.In certain embodiments, the diabetes is type I diabetes.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, selected from rheumatoid arthritis,multiple sclerosis, B-cell chronic lymphocytic leukemia, acutelymphocytic leukemia, hairy cell leukemia, non-Hodgkin's lymphoma,Hodgkin's lymphoma, multiple myeloma, bone cancer, bone metastasis,osteoporosis, diabetes (e.g. type I diabetes), irritable bowel syndrome,Crohn's disease, lupus and renal transplant.

(d) ITK

Interleukin-2 inducible T-cell kinase (“ITK”) is expressed in T cells,mast cells and natural killer cells. It is activated in T cells uponstimulation of the T cell receptor (TCR), and in mast cells uponactivation of the high affinity IgE receptor. Following receptorstimulation in T cells, Lck, a Src tyrosine kinase family member,phosphorylates Y511 in the kinase domain activation loop of ITK (S. D.Heyeck et al., 1997, J. Biol. Chem, 272, 25401-25408). Activated ITK,together with Zap-70 is required for phosphorylation and activation ofPLC-gamma (S. C. Bunnell et al., 2000, J. Biol. Chem., 275, 2219-2230).PLC-gamma catalyzes the formation of inositol 1,4,5-triphosphate anddiacylglycerol, leading to calcium mobilization and PKC activation,respectively. These events activate numerous downstream pathways andlead ultimately to degranulation (mast cells) and cytokine geneexpression (T cells) (Y. Kawakami et al., 1999, J. Leukocyte Biol., 65,286-290).

The role of ITK in T cell activation has been confirmed in ITK knockoutmice. CD4⁺ T cells from ITK knockout mice have a diminishedproliferative response in a mixed lymphocyte reaction or upon Con A oranti-CD3 stimulation. (X. C. Liao and D. R. Littman, 1995, Immunity, 3,757-769). Also, T cells from ITK knockout mice produced little IL-2 uponTCR stimulation resulting in reduced proliferation of these cells. Inanother study, ITK deficient CD4⁺ T cells produced reduced levels ofcytokines including IL-4, IL-5 and IL-13 upon stimulation of the TCR,even after priming with inducing conditions (D. J. Fowell, 1999,Immunity, 11, 399-409).

The role of ITK in PLC-gamma activation and in calcium mobilization wasalso confirmed in the T cells of these knockout mice, which had severelyimpaired IP₃ generation and no extracellular calcium influx upon TCRstimulation (K. Liu et al., 1998, J. Exp. Med. 187, 1721-1727). Suchstudies support a key role for ITK in activation of T cells and mastcells. Thus an inhibitor of ITK would be of therapeutic benefit indiseases mediated by inappropriate activation of these cells.

It has been well established that T cells play an important role inregulating the immune response (Powrie and Coffman, 1993, ImmunologyToday, 14, 270-274). Indeed, activation of T cells is often theinitiating event in immunological disorders. Following activation of theTCR, there is an influx of calcium that is required for T cellactivation. Upon activation, T cells produce cytokines, including IL-2,4, 5, 9, 10, and 13 leading to T cell proliferation, differentiation,and effector function. Clinical studies with inhibitors of IL-2 haveshown that interference with T cell activation and proliferationeffectively suppresses immune response in vivo (Waldmann, 1993,Immunology Today, 14, 264-270). Accordingly, agents that inhibit Tlymphocyte activation and subsequent cytokine production, aretherapeutically useful for selectively suppressing the immune responsein a patient in need of such immunosuppression.

Mast cells play a critical roll in asthma and allergic disorders byreleasing proinflammatory mediators and cytokines. Antigen-mediatedaggregation of Fc.epsilon.RI, the high-affinity receptor for IgE,results in activation of mast cells (D. B. Corry et al., 1999, Nature,402, B18-23). This triggers a series of signaling events resulting inthe release of mediators, including histamine, proteases, leukotrienesand cytokines (J. R. Gordon et al., 1990, Immunology Today, 11,458-464.) These mediators cause increased vascular permeability, mucusproduction, bronchoconstriction, tissue degradation and inflammationthus playing key roles in the etiology and symptoms of asthma andallergic disorders.

Published data using ITK knockout mice suggests that in the absence ofITK function, increased numbers of memory T cells are generated (A. T.Miller et al., 2002 The Journal of Immunology, 168, 2163-2172). Onestrategy to improve vaccination methods is to increase the number ofmemory T cells generated (S. M. Kaech et al., Nature Reviews Immunology,2, 251-262). In addition, deletion of ITK in mice results in reduced Tcell receptor (TCR)-induced proliferation and secretion of the cytokinesIL-2, IL-4, IL-5, IL-10 and IFN-γ (Schaeffer et al, Science 284; 638-641(1999)), Fowell et al, Immunity 11, 399-409 (1999), Schaeffer et al,Nature Immunology 2 (12): 1183-1188 (2001))). The immunological symptomsof allergic asthma are attenuated in ITK−/−mice. Lung inflammation,eosinophil infiltration and mucus production are drastically reduced inITK−/−mice in response to challenge with the allergen OVA (Mueller etal, Journal of Immunology 170: 5056-5063 (2003)). ITK has also beenimplicated in atopic dermatitis. This gene has been reported to be morehighly expressed in peripheral blood T cells from patients with moderateand/or severe atopic dermatitis than in controls or patients with mildatopic dermatitis (Matsumoto et al, International Archives of Allergyand Immunology 129: 327-340 (2002)).

Splenocytes from RLK−/−mice secrete half the IL-2 produced by wild typeanimals in response to TCR engagement (Schaeffer et al, Science 284:638-641 (1999)), while combined deletion of ITK and RLK in mice leads toa profound inhibition of TCR-induced responses including proliferationand production of the cytokines IL-2, IL-4, IL-5 and IFN-γ (Schaeffer etal, Nature Immunology 2 (12): 1183-1188 (2001), Schaeffer et al, Science284: 638-641 (1999)). Intracellular signalling following TCR engagementis effected in ITK/RLK deficient T cells; inositol triphosphateproduction, calcium mobilization, MAP kinase activation, and activationof the transcription factors NFAT and AP-1 are all reduced (Schaeffer etal, Science 284: 638-641 (1999), Schaeffer et al, Nature Immunology 2(12): 1183-1188 (2001)).

Provided compounds are inhibitors of ITK and are therefore useful fortreating one or more disorders associated with activity of ITK. Thus, insome embodiments, the present invention provides a method for treatingan ITK-mediated disorder comprising the step of administering to apatient in need thereof a compound of the present invention, orpharmaceutically acceptable composition thereof.

As used herein, the term “ITK-mediated” disorders or conditions as usedherein means any disease or other deleterious condition in which ITK, ora mutant thereof, is known to play a role. Accordingly, anotherembodiment of the present invention relates to treating or lessening theseverity of one or more diseases in which ITK, or a mutant thereof, isknown to play a role. Specifically, the present invention relates to amethod of treating or lessening the severity of a disease or conditionselected from a mast cell-mediated condition, a basophil-mediateddisorder, an immune or allergic disorder, wherein said method comprisesadministering to a patient in need thereof a compound or compositionaccording to the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with ITK, wherein the disease or condition is animmune disorder, including inflammatory diseases, autoimmune diseases,organ and bone marrow transplant rejection and other disordersassociated with T cell-mediated immune response or mast cell-mediatedimmune response.

In certain embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with ITK, wherein the disease or condition isacute or chronic inflammation, an allergy, contact dermatitis,psoriasis, rheumatoid arthritis, multiple sclerosis, diabetes (e.g. type1 diabetes), inflammatory bowel disease, Guillain-Barre syndrome,Crohn's disease, ulcerative colitis, cancer, graft versus host disease(and other forms of organ or bone marrow transplant rejection) or lupuserythematosus.

In certain embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with ITK, wherein the disease or condition is amast cell driven conditions, a basophil-mediated disorder, reversibleobstructive airway disease, asthma, rhinitis, chronic obstructivepulmonary disease (COPD), peripheral T-cell lymphomas or HIV [also knownas Acquired Immunodeficiency Syndrome (AIDS)]. Such conditions includethose described in Readinger, et al., PNAS 105: 6684-6689 (2008).

(e) JAK Family

The Janus kinases (JAK) are a family of tyrosine kinases consisting ofJAK1, JAK2, JAK3 and TYK2. The JAKs play a critical role in cytokinesignaling. The down-stream substrates of the JAK family of kinasesinclude the signal transducer and activator of transcription (STAT)proteins. JAK/STAT signaling has been implicated in the mediation ofmany abnormal immune responses such as allergies, asthma, autoimmunediseases such as transplant rejection, rheumatoid arthritis, amyotrophiclateral sclerosis and multiple sclerosis as well as in solid andhematologic malignancies such as leukemias and lymphomas. Thepharmaceutical intervention in the JAK/STAT pathway has been reviewed[Frank, Mol. Med. 5: 432-456 (1999) & Seidel, et al, Oncogene 19:2645-2656 (2000)].

JAK1, JAK2, and TYK2 are ubiquitously expressed, while JAK3 ispredominantly expressed in hematopoietic cells. JAK3 binds exclusivelyto the common cytokine receptor gamma chain (yc) and is activated byIL-2, IL-4, IL-7, IL-9, and IL-15.

The proliferation and survival of murine mast cells induced by IL-4 andIL-9 have, in fact, been shown to be dependent on JAK3- and yc-signaling[Suzuki et al, Blood 96: 2172-2180 (2000)].

Cross-linking of the high-affinity immunoglobulin (Ig) E receptors ofsensitized mast cells leads to a release of proinflammatory mediators,including a number of vasoactive cytokines resulting in acute allergic,or immediate (type I) hypersensitivity reactions [Gordon et al, Nature346: 274-276 (1990) & Galli, N. Engl. J. Med., 328: 257-265 (1993)]. Acrucial role for JAK3 in IgE receptor-mediated mast cell responses invitro and in vivo has been established [Malaviya, et al, Biochem.Biophys. Res. Commun. 257: 807-813 (1999)]. In addition, the preventionof type I hypersensitivity reactions, including anaphylaxis, mediated bymast cell-activation through inhibition of JAK3 has also been reported[Malaviya et al, J. Biol. Chem. 274: 27028-27038 (1999)]. Targeting mastcells with JAK3 inhibitors modulated mast cell degranulation in vitroand prevented IgE receptor/antigen-mediated anaphylactic reactions invivo.

A recent study described the successful targeting of JAK3 for immunesuppression and allograft acceptance. The study demonstrated adose-dependent survival of buffalo heart allograft in Wistar Furthrecipients upon administration of inhibitors of JAK3 indicating thepossibility of regulating unwanted immune responses in graft versus hostdisease [Kirken, Transpl. Proc. 33: 3268-3270 (2001)].

IL-4-mediated STAT-phosphorylation has been implicated as the mechanisminvolved in early and late stages of rheumatoid arthritis (RA).Up-regulation of proinflammatory cytokines in RA synovium and synovialfluid is a characteristic of the disease. It has been demostrated thatIL-4-mediated activation of IL-4/STAT pathway is mediated through theJanus kinases (JAK 1 & 3) and that IL-4-associated JAK kinases areexpressed in the RA synovium [Muller-Ladner, et al, J. Immunol. 164:3894-3901 (2000)].

Familial amyotrophic lateral sclerosis (FALS) is a fatalneurodegenerative disorder affecting about 10% of ALS patients. Thesurvival rates of FALS mice were increased upon treatment with a JAK3specific inhibitor. This confirmed that JAK3 plays a role in FALS[Trieu, et al, Biochem. Biophys. Res. Commun. 267: 22-25 (2000)].

Signal transducer and activator of transcription (STAT) proteins areactivated by, among others, the JAK family kinases. Results form arecent study suggested the possibility of intervention in the JAK/STATsignaling pathway by targeting JAK family kinases with specificinhibitors for the treatment of leukemia [Sudbeck, et al., Clin. CancerRes. 5: 1569-1582 (1999)]. JAK3 specific compounds were shown to inhibitthe clonogenic growth of JAK3-expressing cell lines DAUDI, RAMOS, LC1;19, NALM-6, MOLT-3 and HL-60. Inhibition of JAK3 and TYK 2 abrogatedtyrosine phosphorylation of STAT3, and inhibited cell growth of mycosisfungoides, a form of cutaneous T cell lymphoma.

JAK3 has also been implicated in diabetes. A JAK3 inhibitor exhibitedpotent immunomodulatory activity and delayed the onset of diabetes inthe NOD mouse model of autoimmune type 1 diabetes (Cetkovic-Cvrlje, etal. Clin. Immunol. 106: 213-225 (2003)).

According to another embodiment, the invention provides a method fortreating or lessening the severity of a JAK3-mediated disease orcondition in a patient comprising the step of administering to saidpatient a composition according to the present invention.

The term “JAK3-mediated disease”, as used herein means any disease orother deleterious condition in which a JAK3 kinase is known to play arole. Accordingly, another embodiment of the present invention relatesto treating or lessening the severity of one or more diseases in whichJAK3 is known to play a role. Specifically, the present inventionrelates to a method of treating or lessening the severity of a diseaseor condition selected from immune responses such as allergic or type Ihypersensitivity reactions, asthma, autoimmune diseases such astransplant rejection, graft versus host disease, rheumatoid arthritis,diabetes (e.g. type I diabetes), amyotrophic lateral sclerosis, andmultiple sclerosis, neurodegenerative disorders such as familialamyotrophic lateral sclerosis (FALS), as well as in solid andhematologic malignancies such as leukemias and lymphomas, wherein saidmethod comprises administering to a patient in need thereof acomposition according to the present invention.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity ofcancer, an autoimmune disorder, a neurodegenerative or neurologicaldisorder, schizophrenia, a bone-related disorder, liver disease, or acardiac disorder. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the infection, the particular agent, its modeof administration, and the like. Compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

Pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

According to one embodiment, the invention relates to a method ofinhibiting protein kinase activity in a biological sample comprising thestep of contacting said biological sample with a compound of thisinvention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or amutant thereof, activity in a biological sample comprising the step ofcontacting said biological sample with a compound of this invention, ora composition comprising said compound. In certain embodiments, theinvention relates to a method of irreversibly inhibiting ErbB1, ErbB2,ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or a mutant thereof, activityin a biological sample comprising the step of contacting said biologicalsample with a compound of this invention, or a composition comprisingsaid compound.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of protein kinase, or a protein kinase selected from ErbB1,ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or a mutant thereof,activity in a biological sample is useful for a variety of purposes thatare known to one of skill in the art. Examples of such purposes include,but are not limited to, blood transfusion, organ transplantation,biological specimen storage, and biological assays.

Another embodiment of the present invention relates to a method ofinhibiting protein kinase activity in a patient comprising the step ofadministering to said patient a compound of the present invention, or acomposition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting one or more of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase,and/or JAK3, or a mutant thereof, activity in a patient comprising thestep of administering to said patient a compound of the presentinvention, or a composition comprising said compound. According tocertain embodiments, the invention relates to a method of irreversiblyinhibiting one or more of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase,and/or JAK3, or a mutant thereof, activity in a patient comprising thestep of administering to said patient a compound of the presentinvention, or a composition comprising said compound. In otherembodiments, the present invention provides a method for treating adisorder mediated by one or more of ErbB1, ErbB2, ErbB3, ErbB4, aTEC-kinase, and/or JAK3, or a mutant thereof, in a patient in needthereof, comprising the step of administering to said patient a compoundaccording to the present invention or pharmaceutically acceptablecomposition thereof. Such disorders are described in detail herein.

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents, which are normally administered to treatthat condition, may also be present in the compositions of thisinvention. As used herein, additional therapeutic agents that arenormally administered to treat a particular disease, or condition, areknown as “appropriate for the disease, or condition, being treated.”

For example, compounds of the present invention, or a pharmaceuticallyacceptable composition thereof, are administered in combination withchemotherapeutic agents to treat proliferative diseases and cancer.Examples of known chemotherapeutic agents include, but are not limitedto, Adriamycin, dexamethasone, vincristine, cyclophosphamide,fluorouracil, topotecan, taxol, interferons, platinum derivatives,taxane (e.g., paclitaxel), vinca alkaloids (e.g., vinblastine),anthracyclines (e.g., doxorubicin), epipodophyllotoxins (e.g.,etoposide), cisplatin, an mTOR inhibitor (e.g., a rapamycin),methotrexate, actinomycin D, dolastatin 10, colchicine, emetine,trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide,amphotericin, alkylating agents (e.g., chlorambucil), 5-fluorouracil,campthothecin, cisplatin, metronidazole, and Gleevec™, among others. Inother embodiments, a compound of the present invention is administeredin combination with a biologic agent, such as Avastin or VECTIBIX.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with an antiproliferative or chemotherapeutic agent selectedfrom any one or more of abarelix, aldesleukin, alemtuzumab,alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenictrioxide, asparaginase, azacitidine, BCG Live, bevacuzimab,fluorouracil, bexarotene, bleomycin, bortezomib, busulfan, calusterone,capecitabine, camptothecin, carboplatin, carmustine, celecoxib,cetuximab, chlorambucil, cladribine, clofarabine, cyclophosphamide,cytarabine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin,dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicinhydrochloride, dromostanolone propionate, epirubicin, epoetin alfa,erlotinib, estramustine, etoposide phosphate, etoposide, exemestane,filgrastim, floxuridine fludarabine, fulvestrant, gefitinib,gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate,hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib mesylate,interferon alfa-2a, interferon alfa-2b, irinotecan, lenalidomide,letrozole, leucovorin, leuprolide acetate, levamisole, lomustine,megestrol acetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate,methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone,nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel,palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim,pemetrexed disodium, pentostatin, pipobroman, plicamycin, porfimersodium, procarbazine, quinacrine, rasburicase, rituximab, sargramostim,sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen,temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG,thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin,ATRA, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine,zoledronate, or zoledronic acid.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation: treatments for Alzheimer'sDisease such as donepezil hydrochloride (Aricept®) and rivastigmine(Exelon®); treatments for Parkinson's Disease such as L-DOPA/carbidopa,entacapone, ropinrole, pramipexole, bromocriptine, pergolide,trihexephendyl, and amantadine; agents for treating Multiple Sclerosis(MS) such as beta interferon (e.g., Avonex® and Rebif®), glatirameracetate (Copaxone®), and mitoxantrone; treatments for asthma such asalbuterol and montelukast (Singulair®); agents for treatingschizophrenia such as zyprexa, risperdal, seroquel, and haloperidol;anti-inflammatory agents such as corticosteroids, TNF blockers, IL-1 RA,azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and anti-viral agents;agents for treating blood disorders such as corticosteroids,anti-leukemic agents, and growth factors; and agents for treatingimmunodeficiency disorders such as gamma globulin.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with a monoclonal antibody or an siRNA therapeutic.

Those additional agents may be administered separately from an inventivecompound-containing composition, as part of a multiple dosage regimen.Alternatively, those agents may be part of a single dosage form, mixedtogether with a compound of this invention in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a provided compound, anadditional therapeutic agent, and a pharmaceutically acceptable carrier,adjuvant, or vehicle.

The amount of both, an inventive compound and additional therapeuticagent (in those compositions which comprise an additional therapeuticagent as described above)) that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Preferably,compositions of this invention should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of an inventive can beadministered.

In those compositions which comprise an additional therapeutic agent,that additional therapeutic agent and the compound of this invention mayact synergistically. Therefore, the amount of additional therapeuticagent in such compositions will be less than that required in amonotherapy utilizing only that therapeutic agent. In such compositionsa dosage of between 0.01-1,000 μg/kg body weight/day of the additionaltherapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention, or pharmaceutical compositions thereof,may also be incorporated into compositions for coating an implantablemedical device, such as prostheses, artificial valves, vascular grafts,stents and catheters. Vascular stents, for example, have been used toovercome restenosis (re-narrowing of the vessel wall after injury).However, patients using stents or other implantable devices risk clotformation or platelet activation. These unwanted effects may beprevented or mitigated by pre-coating the device with a pharmaceuticallyacceptable composition comprising a kinase inhibitor. Implantabledevices coated with a compound of this invention are another embodimentof the present invention.

5. Probe Compounds

In certain aspects, a compound of the present invention may be tetheredto a detectable moiety to form a probe compound. In one aspect, a probecompound of the invention comprises an irreversible protein kinaseinhibitor of formula I-a or I-b, as described herein, a detectablemoiety, and a tethering moiety that attaches the inhibitor to thedetectable moiety.

In some embodiments, such probe compounds of the present inventioncomprise a provided compound of formula I-a or I-b tethered to adetectable moiety, R^(t), by a bivalent tethering moiety, -T-. Thetethering moiety may be attached to a compound of formula I-a or I-b viaRing A, Ring B, or R¹. One of ordinary skill in the art will appreciatethat when a tethering moiety is attached to R¹, R¹ is a bivalent warheadgroup denoted as R^(1′). In certain embodiments, a provided probecompound is selected from any of formula V-a, V-b, VI-a, VI-b, VII-a, orVII-b:

wherein each of Ring A, Ring B, R¹, m, p, R^(x), R^(y), R^(v), W¹, andW² is as defined above with respect to formulae I-a and I-b, anddescribed in classes and subclasses herein, R¹ is a bivalent warheadgroup, T is a bivalent tethering moiety; and R^(t) is a detectablemoiety.

In some embodiments, R^(t) is a detectable moiety selected from aprimary label or a secondary label. In certain embodiments, R^(t) is adetectable moiety selected from a fluorescent label (e.g., a fluorescentdye or a fluorophore), a mass-tag, a chemiluminescent group, achromophore, an electron dense group, or an energy transfer agent.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and “reporter” and relates to any moiety capableof being detected, e.g., primary labels and secondary labels. A presenceof a detectable moiety can be measured using methods for quantifying (inabsolute, approximate or relative terms) the detectable moiety in asystem under study. In some embodiments, such methods are well known toone of ordinary skill in the art and include any methods that quantify areporter moiety (e.g., a label, a dye, a photocrosslinker, a cytotoxiccompound, a drug, an affinity label, a photoaffinity label, a reactivecompound, an antibody or antibody fragment, a biomaterial, ananoparticle, a spin label, a fluorophore, a metal-containing moiety, aradioactive moiety, quantum dot(s), a novel functional group, a groupthat covalently or noncovalently interacts with other molecules, aphotocaged moiety, an actinic radiation excitable moiety, a ligand, aphotoisomerizable moiety, biotin, a biotin analog (e.g., biotinsulfoxide), a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, a redox-active agent, an isotopicallylabeled moiety, a biophysical probe, a phosphorescent group, achemiluminescent group, an electron dense group, a magnetic group, anintercalating group, a chromophore, an energy transfer agent, abiologically active agent, a detectable label, and any combination ofthe above).

Primary labels, such as radioisotopes (e.g., tritium, ³²P, ³³P, ³⁵S,¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I), mass-tags including, but not limitedto, stable isotopes (e.g., ¹³C, ²H, ¹⁷O, ¹⁸O, ¹⁵N, ¹⁹F, and ¹²⁷I),positron emitting isotopes (e.g., ¹¹C, ¹⁸F, ¹³N, ¹²⁴I, and ¹⁵O), andfluorescent labels are signal generating reporter groups which can bedetected without further modifications. Detectable moities may beanalyzed by methods including, but not limited to fluorescence, positronemission tomography, SPECT medical imaging, chemiluminescence,electron-spin resonance, ultraviolet/visible absorbance spectroscopy,mass spectrometry, nuclear magnetic resonance, magnetic resonance, flowcytometry, autoradiography, scintillation counting, phosphoimaging, andelectrochemical methods.

The term “secondary label” as used herein refers to moieties such asbiotin and various protein antigens that require the presence of asecond intermediate for production of a detectable signal. For biotin,the secondary intermediate may include streptavidin-enzyme conjugates.For antigen labels, secondary intermediates may include antibody-enzymeconjugates. Some fluorescent groups act as secondary labels because theytransfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal.

The terms “fluorescent label”, “fluorescent dye”, and “fluorophore” asused herein refer to moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent labels include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY 650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine(ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3,Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, YO-PRO-1, SYTOXGreen, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3,Fluo-4, fluoro-emerald, YoYo-1 ssDNA, YoYo-1 dsDNA, YoYo-1, SYTORNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX,Spectrum Green, NeuroTrace 500525, NBD-X, MitoTracker Green FM,LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP(post-activation), FlASH-CCXXCC, Azami Green monomeric, Azami Green,green fluorescent protein (GFP), EGFP (Campbell Tsien 2003), EGFP(Patterson 2001), Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bexl, Doxorubicin, LumioGreen, and SuperGlo GFP.

The term “mass-tag” as used herein refers to any moiety that is capableof being uniquely detected by virtue of its mass using mass spectrometry(MS) detection techniques. Examples of mass-tags include electrophorerelease tags such asN-[3-[4′-[(p-Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecoticAcid, 4′-[2,3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) may also beused as mass-tags. Stable isotopes (e.g., ¹³C, ²H, ¹⁷O, ¹⁸O, and ¹⁵N)may also be used as mass-tags.

The term “chemiluminescent group,” as used herein, refers to a groupwhich emits light as a result of a chemical reaction without theaddition of heat. By way of example, luminol(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants likehydrogen peroxide (H₂O₂) in the presence of a base and a metal catalystto produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule whichabsorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

The term “dye,” as used herein, refers to a soluble, coloring substancewhich contains a chromophore.

The term “electron dense group,” as used herein, refers to a group whichscatters electrons when irradiated with an electron beam. Such groupsinclude, but are not limited to, ammonium molybdate, bismuth subnitrate,cadmium iodide, carbohydrazide, ferric chloride hexahydrate,hexamethylene tetramine, indium trichloride anhydrous, lanthanumnitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate,periodic acid, phosphomolybdic acid, phosphotungstic acid, potassiumferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate,silver proteinate (Ag Assay: 8.0-8.5%) “Strong”, silvertetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate,thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranylnitrate, and vanadyl sulfate.

The term “energy transfer agent,” as used herein, refers to a moleculewhich either donates or accepts energy from another molecule. By way ofexample only, fluorescence resonance energy transfer (FRET) is adipole-dipole coupling process by which the excited-state energy of afluorescence donor molecule is non-radiatively transferred to anunexcited acceptor molecule which then fluorescently emits the donatedenergy at a longer wavelength.

The term “moiety incorporating a heavy atom,” as used herein, refers toa group which incorporates an ion of atom which is usually heavier thancarbon. In some embodiments, such ions or atoms include, but are notlimited to, silicon, tungsten, gold, lead, and uranium.

The term “photoaffinity label,” as used herein, refers to a label with agroup, which, upon exposure to light, forms a linkage with a moleculefor which the label has an affinity.

The term “photocaged moiety,” as used herein, refers to a group which,upon illumination at certain wavelengths, covalently or non-covalentlybinds other ions or molecules.

The term “photoisomerizable moiety,” as used herein, refers to a groupwherein upon illumination with light changes from one isomeric form toanother.

The term “radioactive moiety,” as used herein, refers to a group whosenuclei spontaneously give off nuclear radiation, such as alpha, beta, orgamma particles; wherein, alpha particles are helium nuclei, betaparticles are electrons, and gamma particles are high energy photons.

The term “spin label,” as used herein, refers to molecules which containan atom or a group of atoms exhibiting an unpaired electron spin (i.e. astable paramagnetic group) that in some embodiments are detected byelectron spin resonance spectroscopy and in other embodiments areattached to another molecule. Such spin-label molecules include, but arenot limited to, nitryl radicals and nitroxides, and in some embodimentsare single spin-labels or double spin-labels.

The term “quantum dots,” as used herein, refers to colloidalsemiconductor nanocrystals that in some embodiments are detected in thenear-infrared and have extremely high quantum yields (i.e., very brightupon modest illumination).

One of ordinary skill in the art will recognize that a detectable moietymay be attached to a provided compound via a suitable substituent. Asused herein, the term “suitable substituent” refers to a moiety that iscapable of covalent attachment to a detectable moiety. Such moieties arewell known to one of ordinary skill in the art and include groupscontaining, e.g., a carboxylate moiety, an amino moiety, a thiol moiety,or a hydroxyl moiety, to name but a few. It will be appreciated thatsuch moieties may be directly attached to a provided compound or via atethering moiety, such as a bivalent saturated or unsaturatedhydrocarbon chain.

In some embodiments, detectable moieties are attached to a providedcompound via click chemistry. In some embodiments, such moieties areattached via a 1,3-cycloaddition of an azide with an alkyne, optionallyin the presence of a copper catalyst. Methods of using click chemistryare known in the art and include those described by Rostovtsev et al.,Angew. Chem. Int. Ed. 2002, 41, 2596-99 and Sun et al., BioconjugateChem., 2006, 17, 52-57. In some embodiments, a click ready inhibitormoiety is provided and reacted with a click ready -T-R^(t) moiety. Asused herein, “click ready” refers to a moiety containing an azide oralkyne for use in a click chemistry reaction. In some embodiments, theclick ready inhibitor moiety comprises an azide. In certain embodiments,the click ready -T-R^(t) moiety comprises a strained cyclooctyne for usein a copper-free click chemistry reaction (for example, using methodsdescribed in Baskin et al., Proc. Natl. Acad. Sci. USA 2007, 104,16793-16797).

In certain embodiments, the click ready inhibitor moiety is of one ofthe following formulae:

wherein Ring A, Ring B, W¹, W², R^(y), R^(v), p, R^(x), and m are asdefined above with respect to Formula I and described herein, and q is1, 2, or 3.

Exemplary click ready inhibitors include:

In some embodiments, the click ready -T-R^(t) moiety is of formula:

An exemplary reaction in which a click ready inhibitor moiety and aclick ready -T-R^(t) moiety are joined through a [2+3]-cycloadditon isas follows:

In some embodiments, the detectable moiety, R^(t), is selected from alabel, a dye, a photocrosslinker, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, an antibodyor antibody fragment, a biomaterial, a nanoparticle, a spin label, afluorophore, a metal-containing moiety, a radioactive moiety, quantumdot(s), a novel functional group, a group that covalently ornoncovalently interacts with other molecules, a photocaged moiety, anactinic radiation excitable moiety, a ligand, a photoisomerizablemoiety, biotin, a biotin analog (e.g., biotin sulfoxide), a moietyincorporating a heavy atom, a chemically cleavable group, aphotocleavable group, a redox-active agent, an isotopically labeledmoiety, a biophysical probe, a phosphorescent group, a chemiluminescentgroup, an electron dense group, a magnetic group, an intercalatinggroup, a chromophore, an energy transfer agent, a biologically activeagent, a detectable label, or a combination thereof.

In some embodiments, R^(t) is biotin or an analog thereof. In certainembodiments, R^(t) is biotin. In certain other embodiments, R^(t) isbiotin sulfoxide.

In another embodiment, R^(t) is a fluorophore. In a further embodiment,the fluorophore is selected from Alexa Fluor dyes (Alexa Fluor 350,Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680),AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR,BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665),Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, CascadeYellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl,Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, YO-PRO-1, SYTOXGreen, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3,Fluo-4, fluoro-emerald, YoYo-1 ssDNA, YoYo-1 dsDNA, YoYo-1, SYTORNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX,Spectrum Green, NeuroTrace 500525, NBD-X, MitoTracker Green FM,LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP(post-activation), FlASH-CCXXCC, Azami Green monomeric, Azami Green,green fluorescent protein (GFP), EGFP (Campbell Tsien 2003), EGFP(Patterson 2001), Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bexl, Doxorubicin, LumioGreen, or SuperGlo GFP.

As described generally above, a provided probe compound comprises atethering moiety, -T-, that attaches the irreversible inhibitor to thedetectable moiety. As used herein, the term “tether” or “tetheringmoiety” refers to any bivalent chemical spacer including, but notlimited to, a covalent bond, a polymer, a water soluble polymer,optionally substituted alkyl, optionally substituted heteroalkyl,optionally substituted heterocycloalkyl, optionally substitutedcycloalkyl, optionally substituted heterocyclyl, optionally substitutedheterocycloalkylalkyl, optionally substituted heterocycloalkylalkenyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycloalkylalkenylalkyl, an optionallysubstituted amide moiety, an ether moiety, an ketone moiety, an estermoiety, an optionally substituted carbamate moiety, an optionallysubstituted hydrazone moiety, an optionally substituted hydrazinemoiety, an optionally substituted oxime moiety, a disulfide moiety, anoptionally substituted imine moiety, an optionally substitutedsulfonamide moiety, a sulfone moiety, a sulfoxide moiety, a thioethermoiety, or any combination thereof.

In some embodiments, the tethering moiety, -T-, is selected from acovalent bond, a polymer, a water soluble polymer, optionallysubstituted alkyl, optionally substituted heteroalkyl, optionallysubstituted heterocycloalkyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkylalkyl, optionally substitutedheterocycloalkylalkenyl, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substitutedheterocycloalkylalkenylalkyl. In some embodiments, the tethering moietyis an optionally substituted heterocycle. In other embodiments, theheterocycle is selected from aziridine, oxirane, episulfide, azetidine,oxetane, pyrroline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine,pyrazole, pyrrole, imidazole, triazole, tetrazole, oxazole, isoxazole,oxirene, thiazole, isothiazole, dithiolane, furan, thiophene,piperidine, tetrahydropyran, thiane, pyridine, pyran, thiapyrane,pyridazine, pyrimidine, pyrazine, piperazine, oxazine, thiazine,dithiane, and dioxane. In some embodiments, the heterocycle ispiperazine. In further embodiments, the tethering moiety is optionallysubstituted with halogen, —CN, —OH, —NO₂, alkyl, S(O), and S(O)₂. Inother embodiments, the water soluble polymer is a PEG group.

In other embodiments, the tethering moiety provides sufficient spatialseparation between the detectable moiety and the protein kinaseinhibitor moiety. In further embodiments, the tethering moiety isstable. In yet a further embodiment, the tethering moiety does notsubstantially affect the response of the detectable moiety. In otherembodiments, the tethering moiety provides chemical stability to theprobe compound. In further embodiments, the tethering moiety providessufficient solubility to the probe compound.

In some embodiments, a tethering moiety, -T-, such as a water solublepolymer is coupled at one end to a provided irreversible inhibitor andto a detectable moiety, R^(t), at the other end. In other embodiments, awater soluble polymer is coupled via a functional group or substituentof the provided irreversible inhibitor. In further embodiments, a watersoluble polymer is coupled via a functional group or substituent of thereporter moiety.

In some embodiments, examples of hydrophilic polymers, for use intethering moiety -T-, include, but are not limited to: polyalkyl ethersand alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, polyoxyethylene glycol, the latter is also known aspolyethylene glycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof, hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof, terpolymers thereof, mixturesthereof, and derivatives of the foregoing. In other embodiments, a watersoluble polymer is any structural form including but not limited tolinear, forked or branched. In further embodiments, multifunctionalpolymer derivatives include, but are not limited to, linear polymershaving two termini, each terminus being bonded to a functional groupwhich is the same or different.

In some embodiments, a water polymer comprises a poly(ethylene glycol)moiety. In further embodiments, the molecular weight of the polymer isof a wide range, including but not limited to, between about 100 Da andabout 100,000 Da or more. In yet further embodiments, the molecularweight of the polymer is between about 100 Da and about 100,000 Da,including but not limited to, about 100,000 Da, about 95,000 Da, about90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, 30,000 Da,about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da,about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500Da, about 400 Da, about 300 Da, about 200 Da, and about 100 Da. In someembodiments, the molecular weight of the polymer is between about 100 Daand 50,000 Da. In some embodiments, the molecular weight of the polymeris between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of the polymer is between about 1,000 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 5,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 10,000 Da and 40,000 Da. In someembodiments, the poly(ethylene glycol) molecule is a branched polymer.In further embodiments, the molecular weight of the branched chain PEGis between about 1,000 Da and about 100,000 Da, including but notlimited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da.In some embodiments, the molecular weight of a branched chain PEG isbetween about 1,000 Da and about 50,000 Da. In some embodiments, themolecular weight of a branched chain PEG is between about 1,000 Da andabout 40,000 Da. In some embodiments, the molecular weight of a branchedchain PEG is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of a branched chain PEG is betweenabout 5,000 Da and about 20,000 Da. The foregoing list for substantiallywater soluble backbones is by no means exhaustive and is merelyillustrative, and in some embodiments, polymeric materials having thequalities described above are suitable for use in methods andcompositions described herein.

One of ordinary skill in the art will appreciate that when -T-R^(t) isattached to a compound of formula I-a or I-b via the R¹ warhead group,then the resulting tethering moiety comprises the R¹ warhead group. Asused herein, the phrase “comprises a warhead group” means that thetethering moiety formed by —R^(1′)-T- of formula V-a or V-b is eithersubstituted with a warhead group or has such a warhead groupincorporated within the tethering moiety. For example, the tetheringmoiety formed by —R^(1′)-T- may be substituted with an -L-Y warheadgroup, wherein such groups are as described herein. Alternatively, thetethering moiety formed by —R^(1′)-T- has the appropriate features of awarhead group incorporated within the tethering moiety. For example, thetethering moiety formed by —R^(1′)-T- may include one or more units ofunsaturation and optional substituents and/or heteroatoms which, incombination, result in a moiety that is capable of covalently modifyinga protein kinase in accordance with the present invention. Such—R^(1′)-T- tethering moiety are depicted below.

In some embodiments, a methylene unit of an —R^(1′)-T- tethering moietyis replaced by a bivalent -L-Y′— moiety to provide a compound of formulaV-a-iii or V-b-iii:

wherein each of Ring A, Ring B, m, p, R^(x), R^(y), R^(v), W¹, W², T, L,Y′, and R^(t) is as defined above and described in classes andsubclasses herein and Y′ is a bivalent version of the Y group definedabove and described in classes and subclasses herein.

In some embodiments, a methylene unit of an —R^(1′)-T- tethering moietyis replaced by an -L(Y)— moiety to provide a compound of formula V-a-ivor V-b-iv:

wherein each of Ring A, Ring B, m, p, R^(x), R^(y), R^(v), W¹, W², T, L,Y, and R^(t) is as defined above and described in classes and subclassesherein.

In some embodiments, a tethering moiety is substituted with an L-Ymoiety to provide a compound of formula V-a-v or V-b-v:

wherein each of Ring A, Ring B, m, p, R^(x), R^(y), R^(v), W¹, W², T, L,Y, and R^(t) is as defined above and described in classes and subclassesherein.

In certain embodiments, the tethering moiety, -T-, has one of thefollowing structures:

In some embodiments, the tethering moiety, -T-, has the followingstructure:

In other embodiments, the tethering moiety, -T-, has the followingstructure:

In certain other embodiments, the tethering moiety, -T-, has thefollowing structure:

In yet other embodiments, the tethering moiety, -T-, has the followingstructure:

In some embodiments, the tethering moiety, -T-, has the followingstructure:

In some embodiments, -T-R^(t) is of the following structure:

In other embodiments, -T-R^(t) is of the following structure:

In certain embodiments, -T-R^(t) is of the following structure:

In some embodiments, a probe compound of formula V-a, V-b, VI-a, VI-b,VII-a, or VII-b is derived from any compound of Table 5.

In certain embodiments, the probe compound is one of the followingstructures:

It will be appreciated that many -T-R^(t) reagents are commerciallyavailable. For example, numerous biotinylating reagents are availablefrom, e.g., Thermo Scientific having varying tether lengths. Suchreagents include NHS-PEG₄-Biotin and NHS-PEG₁₂-Biotin.

In some embodiments, analogous probe structures to the ones exemplifiedabove are prepared using click-ready inhibitor moieties and click-ready-T-R^(t) moieties, as described herein.

In some embodiments, a provided probe compound covalently modifies aphosphorylated conformation of a protein kinase. In one aspect, thephosphorylated conformation of the protein kinase is either an active orinactive form of the protein kinase. In certain embodiments, thephosphorylated conformation of the protein kinase is an active form ofsaid kinase. In certain embodiments, the probe compound is cellpermeable.

In some embodiments, the present invention provides a method fordetermining occupancy of a protein kinase by a provided irreversibleinhibitor (i.e., a compound of formula I-a or I-b) in a patient,comprising providing one or more tissues, cell types, or a lysatethereof, obtained from a patient administered at least one dose of acompound of said irreversible inhibitor, contacting said tissue, celltype or lysate thereof with a probe compound (i.e., a compound offormula V-a, V-b, VI-a, VI-b, VII-a, or VII-b) to covalent modify atleast one protein kinase present in said lysate, and measuring theamount of said protein kinase covalently modified by the probe compoundto determine occupancy of said protein kinase by said compound offormula I-a or I-b as compared to occupancy of said protein kinase bysaid probe compound. In certain embodiments, the method furthercomprises the step of adjusting the dose of the compound of formula I-aor I-b to increase occupancy of the protein kinase. In certain otherembodiments, the method further comprises the step of adjusting the doseof the compound of formula I-a or I-b to decrease occupancy of theprotein kinase.

As used herein, the terms “occupancy” or “occupy” refer to the extent towhich a protein kinase is modified by a provided covalent inhibitorcompound. One of ordinary skill in the art would appreciate that it isdesirable to administer the lowest dose possible to achieve the desiredefficacious occupancy of the protein kinase.

In some embodiments, the protein kinase to be modified is BTK. In otherembodiments, the protein kinase to be modified is EGFR. In certainembodiments, the protein kinase is JAK. In certain other embodiments,the protein kinase is one or more of ErbB1, ErbB2, or ErbB4. In yetother embodiments, the protein kinase is TEC, ITK, or BMX.

In some embodiments, the probe compound comprises the irreversibleinhibitor for which occupancy is being determined.

In some embodiments, the present invention provides a method forassessing the efficacy of a provided irreversible inhibitor in a mammal,comprising administering a provided irreversible inhibitor to themammal, administering a provided probe compound to tissues or cellsisolated from the mammal, or a lysate thereof, measuring the activity ofthe detectable moiety of the probe compound, and comparing the activityof the detectable moiety to a standard.

In other embodiments, the present invention provides a method forassessing the pharmacodynamics of a provided irreversible inhibitor in amammal, comprising administering a provided irreversible inhibitor tothe mammal, administering a probe compound presented herein to one ormore cell types, or a lysate thereof, isolated from the mammal, andmeasuring the activity of the detectable moiety of the probe compound atdifferent time points following the administration of the inhibitor.

In yet other embodiments, the present invention provides a method for invitro labeling of a protein kinase comprising contacting said proteinkinase with a probe compound described herein. In one embodiment, thecontacting step comprises incubating the protein kinase with a probecompound presented herein.

In certain embodiments, the present invention provides a method for invitro labeling of a protein kinase comprising contacting one or morecells or tissues, or a lysate thereof, expressing the protein kinasewith a probe compound described herein.

In certain other embodiments, the present invention provides a methodfor detecting a labeled protein kinase comprising separating proteins,the proteins comprising a protein kinase labeled by probe compounddescribed herein, by electrophoresis and detecting the probe compound byfluorescence.

In some embodiments, the present invention provides a method forassessing the pharmacodynamics of a provided irreversible inhibitor invitro, comprising incubating the provided irreversible inhibitor withthe target protein kinase, adding the probe compound presented herein tothe target protein kinase, and determining the amount of target modifiedby the probe compound.

In certain embodiments, the probe compound is detected by binding toavidin, streptavidin, neutravidin, or captavidin.

In some embodiments, the probe is detected by Western blot. In otherembodiments, the probe is detected by ELISA. In certain embodiments, theprobe is detected by flow cytometry.

In other embodiments, the present invention provides a method forprobing the kinome with irreversible inhibitors comprising incubatingone or more cell types, or a lysate thereof, with a biotinylated probecompound to generate proteins modified with a biotin moiety, digestingthe proteins, capturing with avidin or an analog thereof, and performingmulti-dimensional LC-MS-MS to identify protein kinases modified by theprobe compound and the adduction sites of said kinases.

In certain embodiments, the present invention provides a method formeasuring protein synthesis in cells comprising incubating cells with anirreversible inhibitor of the target protein, forming lysates of thecells at specific time points, and incubating said cell lysates with aninventive probe compound to measure the appearance of free protein overan extended period of time.

In other embodiments, the present invention provides a method fordetermining a dosing schedule in a mammal for maximizing occupancy of atarget protein kinase comprising assaying a one or more cell types, or alysate thereof, isolated from the mammal, (derived from, e.g.,splenocytes, peripheral B cells, whole blood, lymph nodes, intestinaltissue, or other tissues) from a mammal administered a providedirreversible inhibitor of formula I-a or I-b, wherein the assaying stepcomprises contacting said one or more tissues, cell types, or a lysatethereof, with a provided probe compound and measuring the amount ofprotein kinase covalently modified by the probe compound.

Exemplification

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Compound numbers utilized in the Examples below correspond to compoundnumbers set forth in Table 5, supra.

Example 1 Preparation ofN-(3-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)phenyl) acrylamide I-7

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 120° C., 30 min, MW; B) NMP, 200° C., 10 min, MW; C)NMP, 0° C.-30 min, rt-30 min.

Step-1

A solution of 1 (2.0 g, 0.012 mol), 1,3-phenylenediamine (2.0 g, 0.018mmol), DIPEA (2.33 g, 0.018 mol) in n-BuOH (20 mL) was subjected tomicrowave irradiation at 120° C. for 30 min. The reaction mixture wasthen quenched with water (100 mL), extracted with EtOAc (3×100 mL). Thecombined EtOAc extract was washed with water (100 mL), brine (100 mL),dried over Na₂SO₄ and concentrated under reduced pressure. The residueobtained was further purified column chromatography (SiO₂, 60-120 mesh,EtOAc/CHCl₃: 15/85) gave 3 (1.3 g, 45%) as a dark brown solid.

Step-2

A solution of 3 (1.0 g, 4.27 mmol), 4 (1.5 g, 16.12 mmol) in NMP (10.0mL) was subjected to microwave irradiation (200° C., 10 min). Thereaction mixture was cooled, diluted with water (100 mL) and extractedwith EtOAc (3×100 mL). The combined ethyl acetate extract was washedwith water (100 mL), brine (100 mL), dried over Na₂SO₄ and concentratedunder reduced pressure gave a residue. The crude residue was furtherpurified by column chromatography (SiO₂, CHCl₃/MeOH: 98/2) gave 5 (0.5g, 40.3%) as a light brown solid.

Step-3

To a stirred solution of 5 (200 mg, 0.68 mmol) in NMP (2.0 mL) at 0° C.was added acryloyl chloride (248 mg, 0.2.74 mmol) and the reactionmixture was stirred at 0° C. for 60 min. The reaction mixture was thenstirred with hexane for ½ h and then hexane was removed by decantationfrom the mixture and the residue was quenched with water (10 mL). Theaqueous solution was basified with sat. NaHCO₃ solution and thenextracted with EtOAc (3×10 mL). The combined EtOAc extract was washedwith water (10 mL), brine (10 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The residue obtained was further purified columnchromatography (SiO₂, 230-400, MeOH/CHCl₃: 10/90) gave I-7 (110 mg,46.4%) as a brown solid. ¹H NMR (DMSO-d₆) δ ppm: 2.10 (s, 3H), 5.73 (dd,1.88 & 10.42 Hz, 1H), 6.24 (dd, J=1.88 & 17 Hz, 1H), 6.44 (dd, J=10.08 &16.92 Hz, 1H), 6.78 (t, J=7.36 Hz, 1H), 7.06-7.11 (m, 2H), 7.26 (t,J=8.08 Hz, 1H), 7.38-7.40 (bm, 2H), 7.65 (d, J=8.52 Hz, 2H), 7.88 (s,1H), 7.92 (s, 1H), 8.37 (s, 1H), 8.91 (s, 1H), 10.09 (s, 1H); LCMS: m/e346.8 (M+1).

Example 2 Preparation ofN-(3-(4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)acrylamide I-1

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIEA, n-BuOH, 110° C., 30 min, microwave; B) NMP, 200° C., 10 min,microwave; C) acryloyl chloride, NMP, 0° C.-30 min, rt-30 min.

Step-1

A solution of 1 (0.5 g, 3.35 mmol), m-toluidine (0.36 g, 3.35 mmol),DIEA (0.65 g, 5.0 mmol) in n-BuOH (2.0 mL) was subjected to microwaveirradiation at 110° C. for 30 min. The reaction mixture was thenconcentrated under reduced pressure, quenched with water (5 mL),extracted with EtOAc (3×20 mL). The combined EtOAc extract was washedwith water (5 mL), brine (5 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The residue obtained was further purified columnchromatography (SiO₂, 60-120 mesh, CHCl₃/MeOH: 99/1) gave 3 (0.4 g,54.2%) as a yellow solid.

Step-2

A solution of 3 (0.2 g, 0.91 mmol), 4 (0.2 g, 1.8 mmol) in NMP (2.0 mL)was subjected to microwave irradiation (200° C., 10 min). Then thereaction mixture was cooled, diluted with water (10 mL) and extractedwith CH₂Cl₂ (3×15 mL). The combined CH₂Cl₂ extract was washed with water(5 mL), brine (5 mL), dried over Na₂SO₄ and concentrated under reducedpressure to get a residue. The crude residue was further purified bycolumn chromatography (SiO₂, CHCl₃/MeOH: 98/2) and gave 5 (0.14 g, 53%)as a light yellow solid.

Step-3

To a stirred solution of 5 (0.075 g, 0.25 mmol) in NMP (1.0 mL) at 0° C.was added acryloyl chloride (0.19 g, 2.0 mmoL) and the reaction mixturewas stirred at 0° C. for 30 min followed by stirring at rt for 30 min.The neat reaction mixture was subjected to purification by columnchromatography (neutral Al₂O₃, CHCl₃/MeOH: 98/2) gave I-1 (0.04 g, 45%)as a white solid. ¹H NMR (DMSO-d6) δ ppm: 2.56 (s, 3H), 5.71 (dd, J=2.0& 10.08 Hz, 1H), 6.20-6.25 (m, 2H), 6.45 (dd, J=10.12 & 17.00 Hz, 1H),6.78 (d, J=7.52 Hz, 1H), 7.12-7.19 (m, 2H), 7.31 (d, J=8.44 Hz, 1H),7.46-7.53 (m, 3H), 7.87 (s, 1H), 7.99 (d, J=5.76 Hz, 1H), 9.15 (s, 1H),9.24 (s, 1H), 10.03 (s, 1H); LCMS: m/e 346.4 (M+1).

Example 3 Preparation ofN-(3-(5-methyl-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl) acrylamideI-2

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 110° C., 30 min, MW; B) NMP, 200° C., 15 min, MW; C)acryloyl chloride, NMP, 0° C.-30 min, rt-30 min.

Step-1

A solution of 1 (0.1 g, 0.613 mmol), 2 (0.066 g, 0.613 mmol), DIPEA(0.118 g, 0.919 mmol) in n-BuOH (2.0 mL) was subjected to microwaveirradiation at 110° C. for 90 min. The reaction mixture was cooled,concentrated under reduced pressure and the residue obtained was furtherpurified by column chromatography (SiO₂, Methanol/chloroform mixtures)gave 3 (0.05 g, 34%) as an off white solid.

Step-2

A solution of 3 (0.05 g, 0.213 mmol), 4 (0.046 g, 0.427 mmol) in NMP(2.0 mL) was subjected to microwave irradiation (200° C., 15 min). Thenthe reaction mixture was cooled, diluted with water (15 mL) andextracted with EtOAc (3×15 mL). The combined EtOAc extract was washedwith water (10 mL), brine (10 mL), dried over Na₂SO₄ and concentratedunder reduced pressure gave a residue. The crude residue was furtherpurified by column chromatography (SiO₂, CHCl₃/MeOH: 98/2) gave 5 (0.03g, 46%) as a grey solid.

Step-3

To a stirred solution of 5 (0.025 g, 0.082 mmol) in NMP (0.5 mL) at 0°C. was added acryloyl chloride (0.073 g, 0.821 mmol) and the reactionmixture was stirred at 0° C. for 30 min followed by stirring at rt for30 min. The crude reaction mixture was passed through an alumina column(neutral Al₂O₃, chloroform/methanol mixtures) gave I-2 (0.012 g, 41%) asa pale brown solid. ¹H NMR (DMSO-d₆) δ ppm: 2.10 (s, 3H), 2.27 (s, 3H),5.72 (dd, J=2 & 10.04 Hz, 1H), 6.22 (dd, J=1.96 & 16.92 Hz, 1H), 6.45(dd, J=10.08 & 16.92 Hz, 1H), 6.83 (d, J=7.36 Hz, 1H), 7.09 (t, J=8.06Hz, 1H), 7.17 (t, J=7.78 Hz, 1H), 7.26 (d, J=7.80 Hz, 1H), 7.47 (d,J=1.08 Hz, 1H), 7.53 (s, 1H), 7.58 (d, J=8.60 Hz, 1H), 7.78 (s, 1H),7.88 (s, 1H), 8.15 (s, 1H), 9.01 (s, 1H), 9.99 (s, 1H); LCMS: m/e 360.1(M+1).

Example 4 Preparation ofN-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl) acrylamideI-3

The title compound was prepared according to the schemes, steps andintermediates described below.

A) n-butanol, DIPEA, 110° C., 45 min, MW; B) NMP, 200° C., 10 min, MW;C) NMP, DMAP, 0 OC, 30 min.

Step-1

To a solution of 1 (0.5 g, 3 mmol) in n-butanol (5.0 mL) was added 2(0.64 g, 0.6 mmol), DIPEA (0.116 g, 0.8 mmol) and the reaction mixturewas irradiated under microwave at 110° C. for 45 min. It was cooled,quenched with water (50 mL) and extracted with EtOAc (2×25 mL). Thecombined EtOAc extract was washed with water (25 mL), brine (25 mL),dried over Na₂SO₄ and concentrated under reduced pressure gave 3 (0.45g, 63%) which was taken for the next step without further purification.

Step-2

A solution of 3 (0.45 g, 1.8 mmol) and 4 (0.41 g, 3.7 mmol) in NMP (4.5mL) was subjected to microwave irradiation at 200° C. for 10 min. It wascooled, diluted with water (25 mL) and extracted with EtOAc (3×25 mL).The combined EtOAc extract was washed with water (2×25 mL), brine (25mL), dried over Na₂SO₄ and concentrated under reduced pressure. Theresidue obtained was further purified by column chromatography (SiO₂,60-120, Chloroform/Ethyl acetate: 90/10) gave 5 (0.23 g, 41%) as a lightyellow solid.

Step-3

To a stirred solution of 5 (0.075 g, 0.24 mmol), in NMP (1.5 mL) at 0°C. under N2 atmosphere was added DMAP (0.059 g, 0.48 mmol) and Acryloylchloride (0.064 g, 0.725 mmol) and the reaction mixture was kept at thistemperature for 30 min. It was quenched with water (7.5 mL) andextracted with EtOAc (3×25 mL). The combined EtOAc extract was washedwith 5% Citric acid (10 mL), water (2×10 mL), brine (10 mL), dried overNa₂SO₄ and concentrated under reduced pressure. The crude residue wasfurther purified by column chromatography (Al₂O₃, Chloroform/Methanol:98/2) gave I-3 (0.01 g, 11.3%) as an off white solid. ¹H NMR (DMSO-d₆) δppm: 2.27 (s, 3H), 5.72 (d, J=9.84 Hz, 1H), 6.22 (d, J=16.92 Hz, 1H),6.44 (dd, J=10.2 & 17.02 Hz, 1H), 6.85 (d, J=7.12 Hz, 1H), 7.12-7.19 (m,2H), 7.29 (d, J=7.68 Hz, 1H), 7.43 (d, J=7.92 Hz, 1H), 7.61-7.63 (m,2H), 7.82 (s, 1H), 8.08 (s, 1H), 9.23 (bs, 2H), 10.03 (s, 1H); LCMS: m/e364.2 (M+1).

Example 5 Preparation of(E)-4-(dimethylamino)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)but-2-enamideI-4

The title compound was prepared according to the schemes, steps andintermediates described below.

A) n-butanol, DIPEA, 110° C., 45 min., MW; B) NMP, 200° C., 10 min., MW;C) oxalyl chloride, CH₃CN, ½ h at 0° C., 2 h at 25° C., 5 min at 45° C.;D) NMP, 0° C. to 10° C., 30 min.

Step-1

A solution of 1 (0.5 g, 3.0 mmol), 2 (0.32 g, 3.0 mmol) in n-butanol(5.0 mL) was subjected to microwave irradiation (110° C., 45 min). Itwas cooled, quenched with water (50 mL) and extracted with EtOAc (2×25mL). The combined EtOAc extract was washed with water (25 mL), brine (25mL), dried over Na₂SO₄ and concentrated under reduced pressure gave 3(0.45 g, 63%) which was taken for next step without furtherpurification.

Step-2

A solution of 3 (0.45 g, 1.8 mmol), 4 (0.41 g, 3.7 mmol) in NMP (4.5 mL)was subjected to microwave irradiation (200° C., 10 min). It was cooled,diluted with water (25 mL) and extracted with EtOAc (3×25 mL). Thecombined ethyl acetate extract was washed with water (2×25 mL), brine(25 mL), dried over Na₂SO₄ and concentrated under reduced pressure. Theresidue was further purified by column chromatography (SiO₂,Chloroform/Ethyl acetate: 90/10) gave 5 (0.23 g, 41%) as a light yellowsolid.

Step-3a

To a stirred solution of 6 (0.13 g, 0.80 mmol) in CH₃CN (1.0 mL) wasadded oxalyl chloride (0.122 g, 0.96 mmol) at 0° C. The reaction mixturewas allowed to stir at 0° C. for ½ h and then at RT for 2 h. Finally itwas heated at 45° C. for 5 min, cooled and the reaction mixture wastaken for next step without further purification.

Step-3

To a stirred solution of 5 (0.05 g, 0.16 mmol) in NMP (1.0 mL) was added7 at 0° C. The reaction mixture was stirred at 0° C. for 30 min and at10° C. for 30 min. It was quenched with sat. Sodium bicarbonate soln. (5mL) and extracted with CH₂Cl₂ (3×5 mL). The combined organic extract waswashed with water (1 mL), brine (1 mL) and dried over Na₂SO₄.Concentration under reduced pressure followed by purification by columnchromatography (SiO₂, 230-400, CHCl₃/MeOH, 95/5) gave I-4 (0.02 g,29.4%) as a white solid. ¹H NMR (DMSO-d₆) δ ppm: 2.21 (s, 6H), 2.28 (s,3H), 3.08 (bd, J=5.6 Hz, 2H), 6.29 (d, J=15.60 Hz, 1H), 6.67-6.74 (m,1H), 6.86 (d, J=7.20 Hz, 1H), 7.12-7.20 (m, 2H), 7.27 (d, J=8.00 Hz,1H), 7.43 (d, J=8.00 Hz, 1H), 7.62-7.64 (m, 2H), 7.82 (s, 1H), 8.08 (d,J=3.6 Hz, 1H), 9.23 (s, 1H), 9.24 (s, 1H), 9.96 (s, 1H); LCMS: m/e 421.2(M+1).

Example 6 Preparation ofN-(3-(5-methyl-4-(phenylamino)pyrimidin-2-ylamino)phenyl) acrylamide I-5

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 110° C., 30 min, MW; B) NMP, 200° C., 15 min, MW; C)acryloyl chloride, NMP, 0° C.-30 min, rt-30 min.

Step-1

A solution of 1 (0.1 g, 0.613 mmol), 2 (0.114 g, 1.226 mmol), DIPEA(0.118 g, 0.919 mmol) in n-BuOH (2.0 mL) was subjected to microwaveirradiation at 110° C. for 90 min. The reaction mixture was cooled,concentrated under reduced pressure and the residue was further purifiedby column chromatography (SiO₂, 60-120, Methanol/chloroform: 1/9) gave 3(0.08 g, 59%) as a white solid

Step-2

A solution of 3 (0.08 g, 0.364 mmol), 4 (0.059 g, 0.546 mmol) in NMP(2.0 ml) was subjected to microwave irradiation (200° C., 15 min). Thereaction mixture was cooled, diluted with water (15 mL) and extractedwith EtOAc (3×15 mL). The combined ethyl acetate extract was washed withwater (10 mL), brine (10 mL), dried over Na₂SO₄ and concentrated underreduced pressure gave a residue. The crude residue was further purifiedby column chromatography (SiO₂, 60-120, CHCl₃/MeOH: 98/2) gave 5 (0.06g, 60%) as a light grey solid. ¹H NMR (DMSO-d₆) δ ppm: 2.09 (s, 3H),4.74 (s, 2H), 6.09-6.11 (m, 1H), 6.77-6.85 (m, 2H), 6.91 (t, J=1.72 Hz,1H), 7.02 (t, J=7.36 Hz, 1H), 7.31 (t, J=7.52 Hz, 2H), 7.75 (d, J=7.68Hz, 2H), 7.84 (s, 1H), 8.18 (s, 1H), 8.65 (s, 1H); LCMS: m/e 293.2(M+1).

Step-3

To a stirred solution of 5 (60 mg, 0.205 mmol) in NMP (2.0 mL) at 0° C.was added acryloyl chloride (0.148 g, 1.64 mmol) and the reactionmixture was stirred at 0° C. for 30 min. The neat reaction mixture waspassed through an alumina column (neutral Al₂O₃, chloroform/methanol,99/1) gave I-5 (0.013 g, 18.5%) as an off-white solid. ¹H NMR (DMSO-d₆)δ ppm: 2.11 (s, 3H), 5.72 (dd, J=1.92 & 10.04 Hz, 1H), 6.22 (dd, J=1.92& 16.92 Hz, 1H), 6.45 (dd, J=9.32 & 16.92 Hz, 1H), 7.00 (t, J=7.28 Hz,1H), 7.09 (t, J=8.04 Hz, 1H), 7.23-7.30 (m, 3H), 7.43 (d, J=8.04 Hz,1H), 7.75-7.77 (m, 2H), 7.83 (s, 1H), 7.88 (s, 1H), 8.22 (s, 1H), 9.00(s, 1H), 9.99 (s, 1H); LCMS: m/e 346 (M+1).

Example 7 Preparation ofN-(4-methyl-3-(5-methyl-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)acrylamide I-8

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 120° C., 60 min., MW; A′) (BOC)₂O, MeOH, −10° C., 4 h;B) Pd(OAc)₂, BINAP, Cs₂CO₃, toluene, 110° C., 12 h; C) TFA, CH₂Cl₂, 0°C.-30 min, rt-2 h; D) acryloyl chloride, NMP, 0° C.-30 min, rt-30 min.

Step 1′

To a stirred solution of A (5 g, 0.04 mmol) in MeOH (75 mL) was added(BOC)₂O (11.59 g, 0.050 mmol), slowly at −10° C. The reaction wasstirred at this temperature for 4 h and then reaction mixture wasconcentrated under reduced pressure. The residue obtained was taken inEtOAc (300 mL). It was washed with water (25 mL), brine (25 mL) anddried over Na₂SO₄. Filtration followed by concentration under reducedpressure offered 4 (2.5 g, 27%) as an off-white solid.

Step 1

A solution of 1 (0.5 g, 3.06 mmol), 2 (0.39 g, 3.06 mmol), DIPEA (0.59g, 4.5 mmol) in n-BuOH (5 mL) was subjected to microwave irradiation(120° C., 30 min). The reaction mixture was cooled, solvents removedunder reduced pressure and the residue obtained was quenched with water(5 mL). It was extracted with EtOAc (3×20 mL) and the combined EtOAclayer was washed with water (5 mL), brine (5 mL) and dried over Na₂SO₄.Filtration followed by concentration under reduced pressure offered aresidue which was further purified by column chromatography (SiO₂,60-120, CHCl₃/MeOH: 9/1) gave 3 (0.35 g, 49%) as an off-white solid.

Step 2

A solution of 3 (0.1 g, 0.43 mmol), 4 (0.14 g, 0.64 mmol), Pd(OAc)₂ (10mg, 0.043 mmol), BINAP (0.013 g, 0.021 mmol) and Cs₂CO₃ (0.2 g, 1.06mmol) in degassed toluene (toluene was purged with N₂ for 15 min) wasrefluxed for 12 h under N₂ atmosphere. The reaction mixture was cooledand passed through a short bed of Celite®. The filtrate was diluted withEtOAc (25 mL) and washed with water (5 mL), brine (5 mL) and dried overNa₂SO₄. Filtration followed by concentration under reduced pressureoffered a residue which was further purified by column chromatography(SiO₂, 60-120, CHCl₃/MeOH: 9/1) gave 5 (40 mg, 22%) as an off-whitesolid.

Step-3

To a stirred solution of 5 (0.04 g, 0.095 mmol) in dry CH₂Cl₂ (2 mL) at0° C. was added CF₃COOH (0.2 mL, 5 vol) and the reaction mixture waskept at this temperature for 30 min. It was allowed to come to rt andstir at this temperature for 2 h. It was quenched with ice-cooled water(2 mL), basified with sodium carbonate solution and extracted with EtOAc(2×10 mL). The combined EtOAc extract was washed with water (2 mL),brine (2 mL) and dried over Na₂SO₄. Filtration followed by concentrationunder reduced pressure offered 6 (22 mg, 73%) as a light brown solid.

Step-4

To a stirred solution of 6 (0.2 g, 0.63 mmol) in NMP (4 mL) at 0° C. wasadded acryloyl chloride (0.12 g, 1.25 mmol). The reaction was kept atthis temperature for 30 min and then at rt for 30 min. It was quenchedwith ice-cooled water (2 mL) and extracted with EtOAc (2×10 mL). Thecombined EtOAc extract was washed with water (2 mL), brine (2 mL) anddried over Na₂SO₄. Filtration followed by concentration under reducedpressure offered a residue which was further purified by columnchromatography (SiO₂, 230-400, CHCl₃/MeOH: 9/1) gave I-8 (10 mg, 4%) asa white solid. ¹H NMR (DMSO-d₆) δ ppm: 2.07 (s, 3H), 2.13 (s, 6H), 5.70(dd, J=1.92 & 10.08 Hz, 1H), 6.20 (dd, J=1.96 & 16.88 Hz, 1H), 6.41 (dd,J=10.16 & 16.96 Hz, 1H), 6.69 (d, J=7.36 Hz, 1H), 6.98 (t, J=7.76 Hz,1H), 7.11 (d, J=8.24 Hz, 1H), 7.41 (q, J=9.92 Hz, 1H), 7.49-7.51 (m,2H), 7.73 (s, 1H), 7.80 (s, 1H), 7.97 (s, 1H), 8.16 (s, 1H), 10.00 (s,1H); LCMS: m/e 374 (M+1).

Example 8 Preparation ofN-(3-(4-(3-bromophenylamino)-5-methylpyrimidin-2-ylamino)phenyl)acrylamide I-9

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-butanol, 110° C., 1 h, MW; B) 1.5 N HCl, ethanol, 90° C., 30min., MW; C) acryloyl chloride, NMP, 0° C., 30 min.

Step 1

A solution of 1 (0.5 g, 3.06 mmol), 2 (0.53 g, 3.06 mmol) and DIPEA(0.80 mL, 4.06 mmol) in n-butanol (5 mL) was subjected to microwaveirradiation (110° C., 1 h). The reaction mixture was cooled andconcentrated under reduced pressure gave a residue. The residue taken inEtOAc (5 mL) and washed with NaHCO₃ solution (2 mL), water (2 mL) andwith brine solution (2 mL). Drying over Na₂SO₄ followed by concentrationunder reduced pressure offered crude 3 which was further purified bycolumn chromatography (SiO₂, 60-120, chloroform/methanol, 9/1) gave 3(0.125 g, 13%) as a brown solid.

Step 2

To a solution of 3 (0.15 g, 0.5 mmol) in EtOH (3 mL) was added 4 (0.081g, 0.75 mmol) followed by 1.5 N HCl (0.055 g, 1.5 mmol). The reactionmixture was subjected to microwave irradiation (90° C., 30 min), cooledand concentrated under reduced pressure. The residue obtained was takenin EtOAc (5 mL) and washed with NaHCO₃ solution (2 mL), water (2 mL),and brine (2 mL). It was dried over Na₂SO₄, filtered and concentratedunder reduced pressure gave crude 5. It was further purified by columnchromatography (SiO₂, 60-120, chloroform/methanol, 9/1) gave 5 (0.06 g,32%) as a light brown solid.

Step 3

To a stirred solution of 5 (0.06 g, 0.16 mmol) in NMP (1 mL) was addedacryloyl chloride (0.117 g, 1.29 mmol) at 0° C. The reaction mixture wasallowed to stir at this temperature for 30 min and then taken indichloromethane (2 mL). It was washed with NaHCO₃ solution (1 mL), water(1 mL) and with brine solution (1 mL). It was dried over Na₂SO₄,filtered and concentrated under reduced pressure. The residue wasfurther purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) gave I-9 (0.016 g, 23%) as a pale brown solid.¹H NMR (DMSO-d₆) δ ppm: 2.16 (s, 3H), 5.75 (dd, J=1.72 & 10 Hz, 1H),6.23 (dd, J=1.76 & 16.88, Hz, 1H), 6.45 (dd, J=10.08 & 16.92 Hz, 1H),7.22-7.34 (m, 4H), 7.38 (d, J=8.00 Hz, 1H), 7.63 (d, J=8.08 Hz, 1H),7.77 (s, 1H), 7.82 (s, 1H), 7.93 (s, 1H), 9.68 (s, 1H), 10.26 (s, 1H),10.34 (s, 1H); LCMS: m/e 426 (M+1).

Example 9 Preparation of 3-(4-(2-(cyclopropylsulfonyl)-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-5-methylpyrimidin-2-ylamino)benzenesulfonamideI-10

The title compound was prepared according to the schemes, steps andintermediates described below.

A′) DPPA, Benzyl alcohol, Et₃N, toluene, 110° C., 12 h.; B′) Pd(OH)₂,Ammonium formate, EtOH, reflux, 6 h; A) DIPEA, n-BuOH, 120° C., 1 h.,MW; B) 1.5 N HCl, EtOH, reflux 12 h.; C) Cyclopropylsulphonyl chloride,DIPEA, THF, rt, 12 h.

Steps 1-4 The procedure for synthesizing scaffold 7 is described in theexperimental for Compound I-11 herein.

Step 5

To a stirred solution of 7 (0.05 g, 0.0121 mmol) in THF (4 mL) at 0° C.,was added DIPEA (0.023 g, 0.182 mmol) followed by cyclopropylsulphonylchloride (0.031 g, 0.182 mmol) under N₂ atmosphere. The reaction mixturewas allowed to come to rt and maintained at this temperature for 12 h.It was taken in EtOAc (10 mL), washed with water (5 mL), brine (5 mL)and dried over Na₂SO₄. Filtration followed by concentration underreduced pressure offered a residue which was further purified by columnchromatography (SiO₂, 60-120, pet ether/ethyl acetate, 6/4) gave I-10(0.035 g, 56%) as a yellow solid. ¹H NMR (DMSO-d₆) δ ppm: 0.97-0.1.00(m, 4H), 2.12 (s, 3H), 2.60-2.66 (m, 1H), 2.90 (t, J=5.2 Hz, 2H), 3.52(t, J=6 Hz, 2H), 4.42 (s, 2H), 7.16 (d, J=8.4 Hz, 1H), 7.27 (s, 2H),7.31-7.35 (m, 2H), 7.53 (s, 1H), 7.59 (d, J=8.4 Hz, 1H), 7.92 (s, 1H),8.03-8.04 (m, 2H), 8.45 (s, 1H), 9.40 (s, 1H); LCMS: m/e 515 (M+1).

Example 10 Preparation of3-(4-(2-(2-chloroacetyl)-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-5-methylpyrimidin-2-ylamino)benzenesulfonamideI-11

The title compound was prepared according to the schemes, steps andintermediates described below.

A′) DPPA, Benzyl alcohol, Et₃N, toluene, 110° C., 12 h.; B′) Pd(OH)₂,Ammonium formate, EtOH, reflux, 6 h; A) DIPEA, n-BuOH, 120° C., 1 h.,MW; B) 1.5 N HCl, EtOH, reflux 12 h.; C) Cl—CH₂—COCl, Et₃N, THF, rt, 12h.

Step-1

To a stirred solution of 1 (1.5 g, 5.4 mmol) in toluene (15 mL) wasadded DPPA (2.17 g, 8.11 mmol), Et₃N (1.05 mL, 8.11 mmol) and benzylalcohol (0.876 g, 8.11 mmol) under N₂. The reaction mixture was allowedto reflux for 12 h, cooled and diluted with ethyl acetate (100 mL). Itwas washed with water (5 mL), brine solution (5 mL) and dried overNa₂SO4. It was filtered and concentrated under reduced pressure and theresidue was purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) gave 2 (2.0 g, 97%) as a white solid.

Step-2

To a stirred solution of 2 (2.2 g, 5.75 mmol) in EtOH (25 mL) was addedammonium formate (3.68 g, 57.5 mmol) and the reaction mixture wasrefluxed for 6 h. It was cooled, filtered though a Celite® bed andfiltrate was concentrated under reduced pressure gave 3 (1.3 g, 91%) asa dark brown oil which was used without further purification.

Step-3

A solution of 3 (1.4 g, 5.56 mmol), 4 (0.912 g, 5.56 mmol) and DIPEA(1.077 g, 8.3 mmol) in n-BuOH (15 mL) was subjected to microwaveirradiation at 120° C. for 45 min. The reaction mixture was cooled andconcentrated under reduced pressure. The residue was taken in ethylacetate (20 mL) and washed with water (5 mL) and brine (5 mL). Dryingover Na₂SO₄ followed by concentration under reduced pressure offered aresidue which was purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) gave 5 (1.1 g, 52%) as a cream colored solid.

Step-4

To a stirred solution of 5 (0.25 g, 0.66 mmol) in ethanol (5 mL) wasadded 6 (0.126 g, 0.73 mmol) and catalytic amount of aq.HCl and thereaction mixture was refluxed for 12 h at 100° C. It was cooled, thesolid precipitated was filtered and washed with diethyl ether and driedunder high vacuum gave 7 (0.24 g, 82%) as a light yellow solid.

Step-5

To a stirred solution of 7 (0.2 g, 0.487 mmol) in NMP (5 mL) was addedEt₃N (0.094 g, 0.731 mmol). The solution was cooled to 0° C. andchloroacetylchloride (0.082 g, 0.731 mmol) was added to it. The reactionmixture was allowed to come to rt and stir at this temperature for 12 h.It was quenched with ice cooled water (2 mL) and extracted with ethylacetate (3×5 mL). The combined ethyl acetate extract was washed withbrine solution (2 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The residue obtained was furtherpurified by column chromatography (SiO₂, 60-120, chloroform/methanol,9/1) gave I-11 (0.038 g, 16%) as a light yellow solid. ¹H NMR (DMSO-d₆)δ ppm: 2.11 (s, 3H), 2.77-2.89 (m, 2H), 3.70-3.72 (m, 2H), 4.49 (d,J=2.92 Hz, 2H), 4.63 (d, J=23.56 Hz, 2H), 7.15-7.17 (m, 1H), 7.24 (s,2H), 7.30-7.32 (m, 2H), 7.50-7.65 (m, 2H), 7.91 (s, 1H), 8.04-8.05 (m,2H), 8.27 (s, 1H), 9.31 (s, 1H), LCMS: m/e 486.8 (MH⁺).

Example 11 Preparation ofN-(3-(5-methyl-4-(4-phenoxyphenylamino)pyrimidin-2-ylamino)phenyl)acrylamide I-23

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-butanol, 100° C., 1 h, MW; B) conc.HCl, n-BuOH, 160° C., 20min., MW; C) Acryloyl chloride 0° C., rt, 1 h.

A solution of 1 (0.2 g, 1.2 mmol), 2 (0.12 g, 0.95 mmol) and DIPEA (0.23g, 1.78 mmol) in n-BuOH (2 mL) was subjected to microwave irradiation(100° C. for 1 h). Then the reaction mixture was cooled, concentratedunder reduced pressure and the residue was taken in EtOAc (5 mL). It waswashed with NaHCO₃ solution (2 mL), water (2 mL), brine (2 mL) and thendried over anhydrous Na₂SO₄. Concentrated under reduced pressurefollowed with purification by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) gave 3 (0.11 g, 28.9%) as a light brown solid.

Step-2

To a solution 3 (0.11 g, 0.3 mmol), 4 (0.114 g, 1.05 mmol) in n-butanol(1 mL) was added conc. HCl (1 drop) and the mixture was subjected tomicrowave irradiation (165° C. for 10 min). The reaction mixture wascooled, concentrated under reduced pressure and the residue was taken inEtOAc (5 mL). It was washed with NaHCO₃ solution (2 mL), water (2 mL)and brine (2 mL). Drying over Na₂SO₄ followed by concentration underreduced pressure offered residue which was purified by columnchromatography (SiO₂, 60-120, chloroform/methanol, 9/1) gave 5 (0.08 g,65%) as a brown solid.

Step-3

To a stirred solution 5 (0.015 g, 0.03 mmol) in NMP (1 mL) was addedacryloyl chloride (0.005 g, 0.05 mmol) at 0° C. The reaction mixture wasallowed to come to rt and kept at this temperature for 1 h. It wasdiluted with dichloromethane (2 mL) and washed with NaHCO₃ solution (1mL), water (1 mL) and brine (1 mL). Drying over Na₂SO₄ followed byconcentration under reduced pressure offered a residue which was furtherpurified by column chromatography (SiO₂, 60-120, chloroform/methanol,9/1) gave I-23 (0.004 g, 23%) as a brown solid. 400 MHz, MeOD: δ 2.14(s, 3H), 5.71 (d, J=11.20 Hz, 1H), 6.30-6.44 (m, 2H), 6.94-6.99 (m, 4H),7.07-7.15 (m, 2H), 7.22 (d, J=7.2 Hz, 1H), 7.34-7.36 (m, 3H), 7.63 (d,J=8.8 Hz, 2H), 7.79 (s, 2H); LCMS: m/e 437 (M+1).

Example 12 Preparation ofN-(3-(5-methyl-2-(3-sulfamoylphenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-33

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 120° C., 30 min., MW; B) 1.5 N HCl, Ethanol, 100° C.,12 h; C) NMP, 0° C. to rt, 1 h.

Step-1

A solution of 1 (0.5 g, 3.06 mmol), 1 (0.49 g, 4.59 mmol) and DIPEA(0.59 g, 4.59 mmol) in n-butanol (8 mL) was subjected to microwaveirradiation (120° C., 30 min). It was cooled, quenched with water (5 mL)and extracted with ethyl acetate (3×20 mL). The combined ethyl acetatelayer was washed with brine solution (5 mL), dried over Na₂SO₄, filteredand concentrated under reduced pressure. The residue was furtherpurified by column chromatography (SiO₂, 60-120, chloroform/ethylacetate, 9/1) gave 1 (0.25 g, 34.77%) as a light brown solid.

Step-2

To a stirred solution of 3 (0.1 g, 0.48 mmol), in ethanol (2 mL) wasadded 4 (0.070 g, 0.42 mmol) and catalytic amount of 1.5 N HCl (3drops), then heated to 100° C., for 12 h. Reaction mixture then cooled,solid separated, which was filtered and washed with ether 5 (0.1 g ascrude), which was taken to next step as such.

Step-3

To a stirred solution of 5 (0.1 g, 0.27 mmol) in NMP (2 mL) was addedacryloyl chloride (0.037 g, 0.425 mmol) at 0° C., this was then stirredat room temperature for 1 h, then the reaction mixture was quenched withwater (4 mL) and basified with NaHCO3, this was then extracted withethyl acetate (5 mL), combined organic layer washed with brine solution(1 mL), dried over anhydrous Na₂SO₄, filtered then concentrated, Crudethen purified using preparative HPLC yields I-33 (0.07 g, 6%) as an offwhite solid. ¹H NMR (MeOD) δ ppm: 2.17 (s, 3H), 5.78 (dd, J=2.36 & 9.52Hz, 1H), 6.34-6.48 (m, 2H), 7.26-7.43 (m, 5H), 7.87 (s, 1H), 7.96-8.03(m, 3H); LCMS: m/e 425 (M+1).

Example 13 Preparation ofN-(3-(methyl(5-methyl-2-(phenylamino)pyrimidin-4-yl)amino)phenyl)acrylamide I-34

The title compound was prepared according to the schemes, steps andintermediates described below.

A) Pd(OAC)₂, BINAP, Cs₂CO₃, Toluene, 100° C., 16 h; B) NaH, CH₃I, THF,0° C.-30 min, rt-16 h.; C) Aniline, conc.HCl, Ethanol, 90° C., 60 min;D) H₂, Pd/C, Ethanol, 16 h; E) acryloyl chloride, NMP, 0° C., 1 h.

Step-1

To a stirred solution of 2 (1.0 g, 6.0 mmol), in Toluene (30.0 mL) wasadded 1 (0.84 g, 6.0 mmol), BINAP (0.186 g, 0.3 mmol), Cs₂CO₃ (4.87 g,15.0 mmol). The reaction mixture was degassed by purging N₂ for 15 min.Pd(OAc)₂ (0.134 g, 0.6 mmol) was then added to the reaction mixture andthe reaction mixture was heated at 100° C. for 16 h under N₂ atmosphere.It was then cooled, diluted with Ethyl acetate (30 mL) and filteredthrough Celite®. Filtrate was washed with water (2×25 mL), brine (25mL), dried over Na₂SO₄ and concentrated under reduced pressure. Theresidue obtained was further purified column chromatography (SiO₂,60-120 mesh, Ethylacetete/hexane: 10/90) gave a solid which was washedwith ether gave 3 (0.6 g, 37%) as a light yellow solid.

Step-2

To a stirred mixture of NaH (0.1 g, 2.5 mmol, 60% dispersion in paraffinoil) in dry THF (10.0 mL) was added 3 (0.5 g, 1.89 mmol) at 0° C., andthe reaction mixture was stirred at this temperature for 30 min. CH₃I(0.305 g, 2.15 mmol) was added to it and the reaction was allowed tocome to rt and stir at this temperature for 16 h. The reaction mixturewas diluted with water (25 mL) and extracted with EtOAc (3×25 mL). Thecombined EtOAc extract was washed with water (25 mL), brine (25 mL),dried over Na₂SO₄ and concentrated under reduced pressure gave aresidue. The crude residue was further purified by column chromatography(SiO₂, CHCl₃/MeOH: 99/1) gave 4 (0.12 g, 22.7%) as a light yellow solid.

Step-3

To a solution of 4 (120 mg, 0.431 mmol) in EtOH (2 mL) was addedConc.HCl (0.044 g, 1.2 mmol) and Aniline (0.16 g, 1.72 mmol) and thereaction mixture was heated in a sealed pressure tube at 90° C. for 1 h.The reaction mixture was cooled, solvents removed by concentration underreduced pressure and the residue obtained was diluted with 10% NaHCO₃(10.0 mL). It was extracted with EtOAc (3×15 mL) and the combined EtOAcextract was washed with water (15 mL), brine (15 mL), dried over Na₂SO₄.Concentration under reduced pressure offered a residue which was furtherpurified by column chromatography (SiO₂, CHCl₃/MeOH: 99/1) gave 5 (0.11g, 76%) as a light yellow solid.

Step-4

A solution of 5 (0.110 g, 0.328 mmol), in Ethanol (50 mL)) was added 10%Palladium on charcoal (0.022 g) and the reaction mixture was stirredunder H2 atmosphere (1.5 Kg) at rt for 16 h. It was filtered throughCelite® and concentrated under reduced pressure gave a residue. Theresidue was purified by column chromatography (SiO₂, 60-120,methanol/chloroform: 1/99) gave 6 (0.07 g, 69.9%) as a colorless viscousliquid.

Step-5

To a stirred solution of 6 (0.070 g, 0.23 mmol) in NMP (1.5 mL) at 0° C.was added acryloyl chloride (0.083 g, 0.916 mmol) and the reactionmixture was stirred at 0° C. for 1 h. It was quenched with 10% sodiumbicarbonate solution (15 mL) and the solid precipitated out wasfiltered, washed with cold water (5 mL), hexane (5 mL). The solid wasdried for 2 h under reduced pressure gave I-34 (0.033 g, 40%) as a paleyellow sold. ¹H NMR (DMSO-d₆) δ ppm: δ 1.47 (s, 3H), 3.45 (s, 3H), 5.74(dd, J=Hz, 1H), 6.22 (dd, J=2.0 & 16.98 Hz, 1H), 6.38 (dd, J=10 & 16.94Hz, 1H), 6.85-6.91 (m, 2H), 7.21-7.25 (m, 2H), 7.32 (t, J=8.02 Hz, 1H),7.43-7.47 (m, 2H), 7.77-7.79 (m, 2H), 7.90 (s, 1H), 9.22 (s, 1H), 10.18(s, 1H); LCMS: m/e 360.8 (M+1).

Example 14 Preparation ofN-(3-(5-methyl-2-(3-(prop-2-ynyloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide I-35

The title compound was prepared according to the schemes, steps andintermediates described below.

A) K₂CO₃, CH₃CN, 65° C., 8 h; B) Fe powder, NH₄Cl, MeOH, H₂O, 80° C., 4h; C) 1,3-pheneylendiamine, DIPEA, n-BuOH, 120° C., 30 min, MW; D)Con.HCl, absolute ethanol, 110° C., 2 h; E) NMP, 0° C., 1 h.

Step-1

To a stirred solution of 1a (4 g, 0.0287 mol) and K₂CO₃ (5.6 g, 0.0574mol) in CH₃CN (15 mL) was added propargyl bromide (4.1 g, 0.0345 mol)and the resulting mixture was allowed to reflux for 8 h. The reactionmixture was then cooled, quenched with water and extracted with EtOAc(3×50 mL). The combined EtOAc extract was washed with water (20 mL),brine (20 mL) and dried over Na₂SO₄. Filtration followed byconcentration under reduced pressure furnished 2b as a brownish solidwhich was used without further purification.

Step-2

To a stirred solution of 2b in a mixture of methanol (30 mL) and water(30 mL) was added, NH₄Cl (10.3 g, 0.194 mol) and iron powder (6.8 g,0.121 mol) respectively. Resulting mixture was refluxed at 80° C. for 4h. Reaction mixture was cooled, diluted with methanol and filteredthrough a pad of Celite®. The filtrate was concentrated under reducedpressure and the residue was taken in EtOAc. It was washed with water,brine, dried over Na₂SO₄ and concentrated under reduced pressure gave aresidue. The residue was further purified by column chromatography(SiO2, 60-120, gravity column chromatography, the expected product waseluted with CHCl₃/MeOH: 96/4) gave 3 (3.2 g, 91%) as a brownish solid.

Step-3

A solution of 2, 4-dichloro-5-methyl pyrimidine 1 (0.3 g, 0.0018 mol),1,3-phenylene diamine (0.24 g, 0022 mol), DIPEA (0.35 g, 0.0027 mol) inn-BuOH (3 mL) was subjected to microwave irradiation (120° C., 30 min).The reaction mixture was cooled, quenched with water (15 mL) andextracted with EtOAc (3×15 mL). The combined EtOAc extract was washedwith water (20 mL), brine (20 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The residue obtained was further purified columnchromatography (SiO2, 60-120) gave 2 (0.15 g, 35%) as a brownish solid.

Step-4

2 (0.15 g, 0.006 mol) and 3 (0.37 g, 0.0025 mol) were taken in apressure tube and to it were added abs. EtOH (3 mL) followed by conc.HCl (0.04 g, 0.0012 mol). The tube was tightly screw fitted and washeated at 120° C. for 2 h. The reaction mixture was then cooled,solvents removed under reduced pressure and residue obtained was takenin EtOAc (10 mL). It was washed with water (4 mL), NaHCO₃ (4 mL) andbrine (5 mL). Drying over Na2SO4 followed by concentration under reducedpressure offered a residue which was further purified by columnchromatography (SiO₂, 60-120, gravity column chromatography, expectedcompound getting eluted in CHCl₃/MeOH: 94/6) gave 4 (125 mg, 56%) as alight brown solid.

Step-5

To a stirred solution of 4 (0.1 g, 0.002 mol) in NMP (8 mL) was addedacryloyl chloride (0.1 g, 0.001 mol) drop wise at 0° C. The reaction waskept at this temperature for 10 min and then allowed to come to rt andstir at this temperature for 1.5 h. It was then quenched with 10% sodiumbicarbonate solution (8 mL) and extracted with EtOAc (2×15 mL). Thecombined EtOAc extract was washed with water (10 mL), brine (10 mL)dried over Na₂SO₄ and concentrated under reduced pressure. The residueobtained was purified by column chromatography (SiO2, 60-120, gravitycolumn chromatography, expected compound getting eluted in CHCl₃/MeOH:90/10) gave I-35 (20 mg, 18%) as an off-white solid. ¹H NMR (DMSO-d₆) δppm: 2.11 (s, 3H), 3.51 (s, 1H), 4.61 (s, 2H), 5.74 (d, J=9.08 Hz, 1H),6.25 (d, J=15.84 Hz, 1H), 6.45 (s, 2H), 7.02 (s, 1H), 7.27-7.45 (m, 5H),7.91 (d, J=8.84 Hz, 2H), 8.36 (s, 1H), 8.93 (s, 1H), 10.09 (s, 1H),LCMS: m/e 400 (M+1).

Example 15 Preparation of(E)-4-(dimethylamino)-N-(3-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)phenyl)but-2-enamide I-38

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIEA, n-BuOH, 120° C., 30 min, MW; B) NMP, 200° C., 10 min, MW; C))oxalyl chloride, CH₃CN, 30 min at 0° C., 2 h at 25° C., 5 min at 45° C.,D) NMP, 0° C., 1 h.

Step-1

A solution of 1 (2.0 g, 12 mmol), 2 (2.0 g, 18 mmol), DIPEA (2.33 g, 18mmol) in n-BuOH (20.0 mL) was subjected to microwave irradiation at 120°C. for 30 min. The reaction mixture was then quenched with water (100mL), extracted with EtOAc (3×100 mL). The combined EtOAc extract waswashed with water (100 mL), brine (100 mL), dried over Na₂SO₄ andconcentrated under reduced pressure. The residue obtained was furtherpurified column chromatography (SiO₂, 60-120 mesh, EtOAc/CHCl₃:15/85)gave 3 (1.3 g, 45%) as a dark brown solid.

Step-2

A solution of 3 (1.0 g, 4.27 mmol), 4 (1.5 g, 16.12 mmol) in NMP (10 mL)was subjected to microwave irradiation (200° C., 10 min). Then thereaction mixture was cooled, diluted with water (100 mL) and extractedwith EtOAc (3×100 mL). The combined EtOAc extract was washed with water(100 mL), brine (100 mL), dried over Na₂SO₄ and concentrated underreduced pressure gave a residue. The crude residue was further purifiedby column chromatography (SiO₂, 60-120, CHCl₃/MeOH: 98/2) gave 5 (0.5 g,40.3%) as a light brown solid.

Step-2′

To a stirred solution of 6′ (70 mg, 0.42 mmol) in CH₃CN (1.0 mL) wasadded oxalyl chloride (80 mg, 0.62 mmol) at 0° C. The reaction mixturewas allowed to stir at 0° C. for ½ h and then at rt for 2 h. Finally itwas heated at 45° C. for 5 min, cooled and the reaction mixture wastaken for the next step without further purification.

Step-3

To a stirred solution of 5 (75 mg, 0.12 mmol) in NMP (1 mL) was added 6at 0° C. The reaction mixture was stirred at 0° C. for 1 h, quenchedwith cold water (5 mL), basified with Et₃N and extracted with CH₂Cl₂(3×10 mL). The combined organic extract was washed with water (5 mL),brine (5 mL) and dried over Na₂SO₄. Concentration under reduced pressurefollowed by purification over silica gel (60-120) using 5% methanol inchloroform gave crude compound (20 mg) as a brown gummy solid, which wasagain taken into dichloromethane and stirred with 10% bicarbonatesolution for 30 min, dichloromethane layer separated, dried over Na₂SO₄and concentrated to give I-38 (8 mg, 17%) as a brown solid. ¹H NMR(DMSO-d₆) δ ppm: 2.15 (s, 3H), 2.32 (s, 6H), 3.21 (d, J=5.76 Hz, 2H),6.27 (d, J=15.36 Hz, 1H), 6.84-6.93 (m, 2H), 7.14 (t, J=7.52 Hz, 2H),7.27-7.33 (m, 2H), 7.44 (dd, J=2.04 Hz & 5.08 Hz, 1H), 7.53 (d, J=7.72Hz, 2H), 7.80 (s, 1H), 8.00 (s, 1H); LCMS: m/e 402.8 (M+1).

Example 16 Preparation ofN-(4-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)phenyl) acrylamideI-39

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 110° C., 45 min, MW; B) Conc. HCl, n-BuOH, 150° C., 10min, MW; C) Acryloyl chloride, NMP, 0° C.-30 min, rt-2 h.

Step-1

A solution of 1 (0.4 g, 2.4 mmol), 2 (0.3 g, 2.6 mmol), DIPEA (0.46 g,3.6 mmol) in n-BuOH (10 mL) was subjected to microwave irradiation (110°C., 45 min). The reaction mixture was cooled, quenched with water (20mL) and extracted with EtOAc (3×15 mL). The combined EtOAc extract waswashed with water (20 mL), brine (20 mL), dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by columnchromatography (SiO₂, 60-120, CHCl₃/MeOH: 99/1) gave 3 (350 mg, 62%) asan off-white solid.

Step-2

A solution of 3 (0.2 g, 0.8 mmol), 4 (0.63 g, 6.8 mmol) and con.HCl(0.03 g, 0.8 mmol) in n-BuOH (10 mL) was subjected to microwaveirradiation (150° C., 10 min). Then the reaction mixture was cooled,diluted with water (10 mL), basified with 10% sodium bicarbonatesolution and extracted with EtOAc (3×15 mL). The combined EtOAc extractwas washed with water (15 mL), brine (15 mL), dried over Na₂SO₄ andconcentrated under reduced pressure. The crude residue was purified bycolumn chromatography (SiO₂, 60-120, CHCl₃/MeOH: 97/3) gave 5 (110 mg,47%) as a brown colored gummy solid.

Step-3

To a stirred solution of 5 (0.06 g, 0.2 mmol) in NMP (2 mL) was addedacryloyl chloride (0.03 g, 0.3 mmol) at 0° C. It was allowed to stir atthe same temperature for 20 min and then at rt for 2 h. The reactionmixture was quenched with water, basified with 10% sodium bicarbonatesolution and extracted with EtOAc (3×10 mL). The combined EtOAc layerwas washed with water (10 mL), brine (10 mL), dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by columnchromatography (SiO₂, 60-120) and finally by preparative HPLC gave I-39(10 mg, 16%) as an off-white solid. ¹H NMR (DMSO-d₆) δ ppm: 2.10 (s,3H), 5.71-5.76 (m, 1H), 6.25 (dd, J=2.04 & 16.96 Hz, 1H), 6.45 (dd,J=10.08 & 16.92 Hz, 1H), 6.84 (t, J=7.30 Hz, 1H), 7.14-7.18 (m, 2H),7.62-7.68 (m, 6H), 7.86 (s, 1H), 8.26 (s, 1H), 8.94 (s, 1H), 10.11 (s,1H), LCMS: m/e 346 (M+1).

Example 17 Preparation ofN-(3-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)phenyl) propionamideI^(R)-7

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 120° C., 30 min, MW; B) NMP, 200° C., 10 min, MW; C)6, NMP, 0° C., 60 min.

Step-1

A solution of 1 (2.0 g, 12 mmol), 2 (2.0 g, 18 mmol), DIPEA (2.33 g, 18mmol) in n-BuOH (20.0 mL) was subjected to microwave irradiation at 120°C. for 30 min. The reaction mixture was then quenched with water (100mL), extracted with EtOAc (3×100 mL). The combined EtOAc extract waswashed with water (100 mL), brine (100 mL), dried over Na₂SO₄ andconcentrated under reduced pressure. The residue obtained was furtherpurified column chromatography (SiO₂, 60-120 mesh, EtOAc/CHCl₃: 15/85)gave 3 (1.3 g, 45%) as a dark brown solid.

Step-2

A solution of 3 (1.0 g, 4.27 mmol), 4 (1.5 g, 16.12 mmol) in NMP (10 mL)was subjected to microwave irradiation (200° C., 10 min). Then thereaction mixture was cooled, diluted with water (100 mL) and extractedwith EtOAc (3×100 mL). The combined EtOAc extract was washed with water(100 mL), brine (100 mL), dried over Na₂SO₄ and concentrated underreduced pressure gave a residue. The crude residue was further purifiedby column chromatography (SiO₂, CHCl₃/MeOH: 98/2) gave 5 (0.5 g, 40.3%)as a light brown solid.

Step-3

To a stirred solution of 5 (75 mg, 0.25 mmol) in NMP (1.0 mL) at 0° C.was added propanoyl chloride (6) (72 mg, 0.75 mmol) and the reactionmixture was stirred at 0° C. for 60 min. The reaction mixture was thenquenched with water (5 mL), basified with Et₃N and extracted with EtOAc(3×10 mL). The combined EtOAc extract was washed with water (10 mL),brine (10 mL), dried over Na₂SO₄ and concentrated under reducedpressure. The residue obtained was further purified columnchromatography (SiO₂, 230-400, methanol/chloroform: 2/98) gave I^(R)-7(0.025 g, 28.73%) as an off white solid. ¹H NMR (DMSO-d₆) δ ppm: 1.08(t, J=7.6 Hz, 3H), 2.11 (s, 3H), 2.31 (q, J=7.6 Hz, 2H), 6.81 (t, J=7.2Hz, 1H), 7.11 (t, J=8 Hz, 2H), 7.21-7.25 (m, 1H), 7.31 (d, J=8.40 Hz,1H), 7.36 (d, J=8.00 Hz, 1H), 7.66 (d, J=8.40 Hz, 2H), 7.86 (s, 1H),7.89 (s, 1H), 8.35 (s, 1H), 8.93 (s, 1H), 9.81 (s, 1H); LCMS: m/e 348.3(M+1).

Example 18 Preparation ofN-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl) acrylamideI-56

The title compound was prepared according to the schemes, steps andintermediates described below.

A) acryloyl chloride, Et₃N, DMF, rt, 12 h

Step-1

To a stirred solution of 1 (0.15 g, 0.54 mmol) and Et₃N (0.11 g, 1.08mmol) in DMF (1 mL) at 0° C. was added acryloyl chloride (0.09 g, 1.08mmol), drop-wise, under N₂ atmosphere. The reaction mixture was allowedto come to rt and stirred further 12 h. It was then quenched withice-cold water (2 mL) and extracted with EtOAc (2×15 mL). The combinedEtOAc extract was washed with brine (2 mL), dried over Na₂SO₄ andconcentrated under reduced pressure to get a crude residue. The residuewas further purified by preparative HPLC and gave I-56 (0.060 g, 33%) asa pale yellow solid. ¹H NMR (DMSO-d₆) δ ppm: 2.19 (s, 3H), 5.72 (dd, J=2& 10.08 Hz, 1H), 6.22 (dd, J=2 & 16.92 Hz, 1H), 6.45 (dd, J=10 & 17 Hz,1H), 7.16 (d, J=8.36 Hz, 1H), 7.32 (dd, J=1.92 & 8.16 Hz, 1H), 7.42 (d,J=5.12 Hz, 1H), 7.50-7.53 (m, 1H), 7.95 (d, J=1.68 Hz, 1H), 8.45 (dd,J=6.16 & 8.16 Hz, 1H), 8.49 (d, J=5.16 Hz, 1H), 8.68 (dd, J=1.56 & 4.76Hz, 1H), 8.95 (s, 1H), 9.25 (d, J=1.56 Hz, 1H), 10.08 (s, 1H); LCMS: m/e332.4 (M+1).

Example 19

General method for preparing compounds having an enone-containingwarhead, e.g.,3-methyl-1-(3-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)phenyl)but-2-en-1-oneI-47

The title compound is prepared according to the schemes, steps andintermediates described below. It is also appreciated by one skilled inthe art that I-47 is an exemplary compounds having enone-containingwarheads, and that other compounds having enone-containing warheads canbe synthesized in a substantially similar manner according to theschemes, steps and corresponding intermediates described below.

Compounds 1 and 2 are coupled in the presence of triethylamine to yieldcompound 3. Compound 3 is treated with analine at elevated temperatureto yield compound 4. Saponification of Compound 4 with potassiumhydroxide yields acid compound 5, which is coupled toN—O-dimethylhydroxylamine using EDC to yield compound 6. Treatment ofCompound 6 at low temperature yields exemplary compound I-47.

Example 20 Preparation ofN-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-182

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, DIPEA, THF, reflux; B) 4, t-amyl alcohol, HOAc, reflux; C) TFA,DCM; D) 7, DIPEA, THF, −10° C.

Step-1

1 (800 mg, 4.8 mmoL), 2 (996 mg, 4.8 mmoL) and Hunig's base (948 uL,5.75 mmoL) were dissolved in THF (20 mL). The reaction mixture washeated at reflux overnight. After cooling, partitioned betweenwater/brine (10 mL), agitated and separated the layers. Dried organicphase over sodium sulfate, and the solvent was removed via rotaryevaporation. Titration with EtOAc and Heptane gave after filtration awhite solid, 1 g. LC/MS (RT=2.03/(M+1)) 339.1.

Step-2

3 (800 mg, 2.37 mmoL) and 4 (576 mg, 2.84 mmoL) were suspended intert-amyl alcohol (14 mL) and acetic acid (5 drops). Heated to refluxfor 4 h. After cooling, solvent was removed via rotary evaporation. Thedark oil was partitioned between water/brine and THF (10 mL each),agitated, and separated layers and dried organic phase over sodiumsulfate. The solvent was removed via rotary evaporation to afford apurple solid, 0.55 g. LC/MS (RT=2.997/(M+1)) 470.2. Additional 150 mg ofproduct minus the (BOC) protecting group crystallized from the aqueouslayer.

Step-3

To a solution of 6 (550 mg, 1.17 mmol) in DCM (20 mL) was added TFA (2mL). Stirred for 30 min at rt for 4 h; removed solvent via rotaryevaporation and partitioned oil with cold (0° C.) saturated sodiumbicarbonate (10 mL) and EtOAc (10 mL), agitated and separated layers.Organic phase was dried over sodium sulfate and the solvent was removedvia rotary evaporation to give a dark oil. Flash chromatography using20%-100% Heptane/EtOAc gradient using combiflash system gave 309 mg of alight pink solid. LC/MS (RT=2.78/(M+1)) 370.2.

Step-4

A solution of 6 (309 mg, 0.84 mmol) in THF (10 mL) was cooled in awater/ice-MeOH bath (−10° C.). To this was added 7 (71 μL, 0.88 mmoL),stirred for 10 min, then added Hunig's base (145 uL, 0.88 mmoL), andstirred for 10 min. Partitioned between water/brine (10 mL), agitatedand separated the layers. Dried organic phase over sodium sulfate. Thesolvent was removed via rotary evaporation and triturated with diethylether to afford after filtration 285 mg (80%) of an off-white solid.LC/MS (RT=2.79/(M+H)) 424.2.

Example 21 Preparation ofN-(3-(2-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-86

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-chloro-4-(pyridine-2-ylmethoxy)aniline in the place of 4 in Step 2.LC/MS (RT=2.87/(M+H)) 491.1.

Example 22 Preparation ofN-(3-(5-fluoro-2-(4-(2-(2-oxopyrrolidin-1-yl)ethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-92

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using1-(2-(4-aminophenoxy)ethyl)pyrrolidin-2-one in the place of 4 in Step 2.LC/MS (RT=2.718/(M+H)) 477.1.

Example 23 Preparation ofN-(3-(5-fluoro-2-(4-(1-hydroxy-2-methylpropan-2-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-93

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using2-(4-aminophenoxy)-2-methylpropan-1-ol in the place of 4 in Step 2.LC/MS (RT=2.724/(M+H)) 438.1.

Example 24 Preparation ofN-(3-(5-fluoro-2-(6-isopropoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-172

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using6-isopropoxypyridin-3-amine in the place of 4 in Step 2. LC/MS(RT=2.878/(M+H)) 409.2.

Example 25 Preparation ofN-(3-(5-fluoro-2-(2-oxoindolin-5-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-181

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using 5-aminoindolin-2-one inthe place of 4 in Step 2. LC/MS (RT=2.617/(M+H)) 405.1.

Example 26 Preparation ofN-(2-chloro-5-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-108

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using tert-butyl5-amino-2-chlorophenylcarbamate in the place of 2 in Step 1. LC/MS(RT=2.852/(M+H)) 458.1.

Example 27 Preparation ofN-(2-chloro-5-(5-fluoro-2-(6-isopropoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-107

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using tert-butyl5-amino-2-fluorophenylcarbamate in the place of 2 in Step 1 and6-isopropoxypyridin-3-amine in the place of 4 in Step 2. LC/MS(RT=2.938/(M+H)) 443.1.

Example 28 Preparation ofN-(2-fluoro-5-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-87

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using tert-butyl5-amino-2-fluorophenylcarbamate in the place of 2 in Step 1. LC/MS(RT=2.797/(M+H)) 442.0.

Example 29 Preparation ofN-(3-(5-fluoro-2-(4-((1-methylpiperidin-4-yl)methoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-90

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, DIPEA, THF, reflux; B) 4, Pd(OAc)₂, X-Phos, CsCO₃, dioxane,reflux, 12 h; C) TFA, DCM; D) 7, DIPEA, THF, −10° C.

Step-1

1 (800 mg, 4.8 mmoL), 2 (996 mg, 4.8 mmoL) and Hunig's base (948 uL,5.75 mmoL) were dissolved in THF (20 mL). The reaction mixture washeated at reflux overnight. After cooling, partitioned with water/brine(10 mL), agitated and separated the layers. Dried organic phase oversodium sulfate and the solvent was removed via rotary evaporation.Titration with EtOAc and Heptane gave after filtration a white solid, 1g. LC/MS (RT=2.03/(M+1)) 339.1.

Step-2

3 (205 mg, 0.61 mmoL) and 4 (150 mg, 0.73 mmoL) was dissolved in dioxane(4 mL). Degassed the solution for 1 min. Palladium acetate (20 mg, 5 moL%), X-Phos ligand (35 mg, 10 moL %) and CsCO₃ (325 mg, 1.2 mmoL) wereadded in that order. Degassed the suspension for 1 min and under argonatmosphere the mixture was heated to reflux for 12 h. After cooling,solvent was removed via rotary evaporation. The dark oil was partitionedbetween water/brine and EtOAc (5 mL each), agitated, filtered offprecipitate and separated layers of the filtrate. Dried organic phaseover sodium sulfate. The solvent was removed via rotary evaporation togive a dark oil. Flash chromatography using 0-30% gradient ofHeptane/EtOAc afforded light yellow oil. LC/MS (RT=3.043/(M+1)) 523.2.

Step-3

To a solution of 5 (144 mg, 0.27 mmol) in DCM (10 mL) was added TFA (1mL). Stirred for 30 min at rt for 12 h; removed solvent via rotaryevaporation and partitioned oil with cold (0° C.) saturated sodiumbicarbonate (5 mL) and EtOAc (5 mL), agitated and separated layers.Organic phase was dried over sodium sulfate and the solvent was removedvia rotary evaporation to give light yellow foam. LC/MS (RT=2.723/(M+1))423.1.

Step-4

A solution of 6 (105 mg, 0.25 mmol) in THF (3 mL) was cooled inwater/ice-MeOH bath (−10° C.). To this was added 7 (21 μL, 0.26 mmoL),stirred for 10 min, then added Hunig's base (51 μL, 0.26 mmoL), andstirred for 10 min. Partitioned with water/brine (5 mL), agitated andseparated the layers. Dried organic phase over sodium sulfate. Thesolvent was removed via rotary evaporation afford a light yellow foam.LC/MS (RT=2.726/(M+H)) 477.1.

Example 30 Preparation ofN-(3-(5-fluoro-2-(6-((2-methoxyethyl)(methyl)amino)pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-77

The title compound was prepared according to the schemes, steps andintermediates described in Example 29, by usingN²-(2-methoxyethyl)-N²-methylpyridine-2,5-diamine in the place of 4 inStep 2. LC/MS (RT=2.739/(M+H)) 438.1.

Example 31 Preparation of1-(6-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)-2H-benzo[b][1,4]oxazin-4(3H)-yl)prop-2-en-1-oneI-194

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, DIPEA, THF, reflux; B) 4, HOAc, tert-amyl alcohol, reflux, 12 h;C) TFA, DCM; D) 7, DIPEA, DCM, NMP, −10° C.

Step-1

1 (186 mg, 1.1 mmoL), 2 (280 mg, 1.1 mmoL) and Hunig's base (220 μL, 1.3mmoL) were dissolved in THF (6 mL). The reaction mixture was heated atreflux overnight. After cooling, partitioned with water/brine (6 mL),agitated and separated the layers. Dried organic phase over sodiumsulfate and the solvent was removed via rotary evaporation to give a tansolid. LC/MS (RT=3.008/(M+1)) 381.1.

Step-2

3 (215 mg, 0.56 mmoL) and 4 (83 mg, 0.66 mmoL) was suspended intert-amyl alcohol (6 mL) and acetic acid (3 drops). Heated to reflux for12 h. After cooling, solvent was removed via rotary evaporation. Thedark oil was partitioned between water/brine and EtOAc (5 mL each),agitated, and separated layers and dried organic phase over sodiumsulfate. The solvent was removed via rotary evaporation to afford anoil. Flash chromatography using 30-70% gradient of heptane/ethyl acetateon combiflash system gave a tan solid. LC/MS (RT=2.011/(M+1)) 469.2.

Step-3

To a solution of 5 (200 mg, 0.43 mmol) in DCM (10 mL) was added TFA (1mL). Stir for 30 min at rt for 12 h; removed solvent via rotaryevaporation and partitioned oil between cold (0° C.) saturated sodiumbicarbonate (5 mL) and EtOAc (5 mL), agitated and separated layers.Organic phase was dried over sodium sulfate and the solvent was removedvia rotary evaporation to give a pink solid. LC/MS (RT=2.782/(M+1))369.1.

Step-4

A solution of 6 (150 mg, 0.41 mmol) in DCM (2 mL) and NMP (0.5 mL) wascooled in water/ice-MeOH bath (−10° C.). To this was added 7 (34 μL,0.43 mmoL), stirred for 10 min, then added Hunig's base (70 μL, 0.43mmoL), and stirred for 10 min. Partitioned between water/brine (5 mL),agitated and separated the layers. Dried organic phase over sodiumsulfate. Purified directly via flash chromatography using 20-80%gradient of heptane/ethyl acetate to give a pink solid. LC/MS(RT=2.8/(M+H)) 423.1.

Preparation of1-(6-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)-2H-benzo[b][1,4]oxazin-4(3H)-yl)prop-2-en-1-oneI-141

The title compound was prepared according to the schemes, steps andintermediates described in Example 31, by using4-(2-methoxyethoxy)aniline in the place of 4 in Step 2. LC/MS(RT=2.845/(M+H)) 466.2.

Example 33 Preparation of1-(6-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)indolin-1-yl)prop-2-en-1-oneI-166

The title compound was prepared according to the schemes, steps andintermediates described in Example 31, by using tert-butyl6-aminoindoline-1-carboxylate in the place of 2 in Step 1. LC/MS(RT=2.825/(M+H)) 407.1.

Example 34 Preparation of1-(5-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)isoindolin-2-yl)prop-2-en-1-oneI-165

The title compound was prepared according to the schemes, steps andintermediates described in Example 31, by using tert-butyl5-aminoisoindoline-1-carboxylate in the place of 2 in Step 1. LC/MS(RT=2.751/(M+H)) 407.1.

Example 35 Preparation of1-(6-(4-(3-chlorophenylamino)-5-fluoropyrimidin-2-ylamino)-2H-benzo[b][1,4]oxazin-4(3H)-yl)prop-2-en-1-oneI-149

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, DIPEA, THF, reflux; B) 4, HOAc, tert-amyl alcohol, reflux, 12 h;C) TFA, DCM; D) 7, DIPEA, THF, −10° C.

Step-1

1 (484 mg, 2.9 mmoL), 2 (305 mg, 2.9 mmoL) and Hunig's base (526 μL, 3.5mmoL) were dissolved in THF (10 mL). The reaction mixture was heated atreflux overnight. After cooling, partitioned between water/brine (10mL), agitated and separated the layers. Dried organic phase over sodiumsulfate and the solvent was removed via rotary evaporation. Flashchromatography using a gradient of 0-30% heptane/ethyl acetate oncombiflash system gave a white solid. LC/MS (RT=2.03/(M+1)) 339.1.

Step-2

3 (150 mg, 0.58 mmoL) and 4 (175 mg, 0.7 mmoL) were suspended intert-amyl alcohol (8 mL) and acetic acid (3 drops). Heated to reflux for12 h. After cooling, solvent was removed via rotary evaporation. Thedark oil was partitioned between water/brine and EtOAC (5 mL each),agitated, and separated layers and dried organic phase over sodiumsulfate. The solvent was removed via rotary evaporation to afford a darkoil. Flash chromatography using a gradient of 0-25% heptane/ethylacetate on combiflash system gave a white solid. LC/MS (RT=2.997/(M+1))470.2.

Step-3

To a solution of 5 (180 mg, 0.38 mmol) in DCM (10 mL) was added TFA (1mL). Stirred for 30 min at rt for 4 h; removed solvent via rotaryevaporation and partitioned oil between cold (0° C.) saturated sodiumbicarbonate (5 mL) and EtOAc (5 mL), agitated and separated layers.Organic phase was dried over sodium sulfate and the solvent was removedvia rotary evaporation to give light yellow solid. LC/MS(RT=2.723/(M+1)) 423.1.

Step-4

A solution of 6 (150 mg, 0.4 mmol) in THF (3 mL) was cooled inwater/ice-MeOH bath (−10° C.). To this was added 7 (34 μL, 0.42 mmoL),stirred for 10 min, then added Hunig's base (70 μL, 0.42 mmoL), andstirred for 10 min. Partitioned between water/brine (5 mL), agitated andseparated the layers. Dried organic phase over sodium sulfate. Thesolvent was removed via rotary evaporation to afford a light yellowsolid. Flash chromatography using gradient of 10-50% heptane/ethylacetate on combiflash system gave a white solid. LC/MS (RT=2.945/(M+H))426.

Example 36 Preparation of5-(2-(4-acryloyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-ylamino)-5-fluoropyrimidin-4-ylamino)indolin-2-oneI-130

The title compound was prepared according to the schemes, steps andintermediates described in Example 35, by using 5-aminoindolin-2-one inthe place of 2 in Step 1. LC/MS (RT=2.673/(M+H)) 447.1.

Example 37 Preparation of4-(3-acrylamidophenylamino)-2-(phenylamino)pyrimidine-5-carboxamideI-230

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, NEt₃, DCM, 0° C. to rt; B) 4, DIPEA, THF, rt, 12 h; C) 6, DIPEA,t-amyl alcohol, reflux, 4 h; D) TFA, DCM, rt; E) 7, NEt₃, THF, 0° C.; F)TFA, TfOH, DCM, rt.

Step-1

1 (500 mg, 2.4 mmoL, prepared from 2,4-dihydroxypyrimidine-5-carboxylicacid according to J. Med. Chem. 50: 591 (2007) and US 2007/0072851) wasdissolved in DCM (10 mL) and chilled in an ice/water bath (0° C.). 2(309 μL, 2.4 mmoL) was added and the mixture stirred for 10 min.Triethylamine (365 μL, 2.6 mmol) was added and the mixture was allowedto warm to rt and stir for 30 min. The solvent was reduced in volume viarotary evaporation and directly purified by flash chromatography using agradient of 0-30% heptane/ethyl acetate on combiflash system to give awhite solid. LC/MS (RT=2.789/(M+1)) 312.

Step-2

3 (170 mg, 0.55 mmoL), 4 (113 mg, 0.55 mmoL) and Hunig's base (108 μL,0.65 mmoL) were dissolved in THF (6 mL). Stirred at rt for 12 h.Partitioned between water/brine, agitated, and separated layers anddried organic phase over sodium sulfate. The solvent was removed viarotary evaporation to afford after titration with EtOAc a white solid.LC/MS (RT=3.123/(M+1)) 484.

Step-3

5 (230 mg, 0.48 mmol), 6 (126 μL, 1.4 mmoL) and Hunig's base (94 μL,0.57 mmoL) is dissolved in t-amyl alcohol (6 mL). Heat to reflux for 4h, cool and water was added to the solid mass. Agitated, filtered anddried to give a white solid. LC/MS (RT=3.182/(M+1)) 541.2.

Step-4

7 (180 mg, 0.33 mmol) was suspended in DCM (10 mL) and treated with TFA(1 mL). Stirred overnight at rt. Diluted with DCM (40 mL) and washedwith NaOH (1N, 25 mL). Agitated, precipitate formed, filtered and dry togive a white solid. LC/MS (RT=2.934/(M+1)) 441.1.

Step-5

A suspension of 8 (130 mg, 0.29 mmol) in THF (6 mL) was cooled inwater/ice (0° C.). To this was added 9 (25 μL (plus additional 5 μL),0.38 mmoL (total)), then added triethyl amine (43 μL (plus additional 11μL), 0.38 mmoL(total)), and stirred for a total time of 1 h. Water wasadded, agitated, filtered off remaining precipitate and discarded. Thefiltrate was dried over sodium sulfate. The solvent was removed viarotary evaporation to afford a yellow solid. Flash chromatography usinga gradient of 0-25% heptane/ethyl acetate on combiflash system gave awhite solid. LC/MS (RT=2.964/(M+H)) 495.1.

Step-6

To a suspension of I-231 (30 mg, 0.061 mmol) in DCM (4 mL) was added TFA(200 L) and triflic acid (68 μL, 0.61 mmoL). Stirred at rt 1 h. Removedsolvent under reduce pressure via rotary evaporation and partitionedwith cold (0° C.) saturated sodium bicarbonate (10 mL) and EtOAc (10mL), agitated and separated layers. Dried organic layer over sodiumsulfate and the solvent was removed via rotary evaporation to affordafter titration with diethyl ether a white solid. LC/MS (RT=2.715/(M+H))375.1.

Example 38 Preparation of4-(3-acrylamidophenylamino)-N-phenyl-2-(phenylamino)pyrimidine-5-carboxamideI-222

The title compound was prepared according to the schemes, steps andintermediates described in Example 37, by using aniline in the place of2 in Step 1 and omitting Step 6. LC/MS (RT=2.991/(M+H)) 451.2.

Example 39 Preparation of4-(3-acrylamidophenylamino)-N-cyclopropyl-2-(phenylamino)pyrimidine-5-carboxamideI-221

The title compound was prepared according to the schemes, steps andintermediates described in Example 37, by using cyclopropylamine in theplace of 2 in Step 1 and omitting Step 6. LC/MS (RT=2.838/(M+H)) 415.2.

Example 40 Preparation of4-(3-acrylamidophenylamino)-2-(3-methoxyphenylamino)pyrimidine-5-carboxamideI-210

The title compound was prepared according to the schemes, steps andintermediates described in Example 37, by using 3-methoxyaniline in theplace of 6 in Step 3. LC/MS (RT=2.743/(M+H)) 405.1.

Example 41 Preparation of4-(3-acrylamidophenylamino)-2-(6-methoxypyridin-3-ylamino)pyrimidine-5-carboxamide I-209

The title compound was prepared according to the schemes, steps andintermediates described in Example 37, by using 6-methoxypyridin-3-aminein the place of 6 in Step 3. LC/MS (RT=2.657/(M+H)) 406.2.

Example 42 Preparation of1-{6-[5-Acetyl-2-(6-methoxy-pyridin-3-ylamino)-pyrimidin-4-ylamino]-2,3-dihydro-benzo[1,4]oxazin-4-yl}-propenoneI-170

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, DIPEA, THF, 70° C., 16 h; B) (a) 4, PdCl₂(PPh₃)₂, DMF, 70° C.; (b)1N HCl, acetone, 60° C., 15 min; C) HCl/dioxane, DCM; D) 7, DIPEA, NMP,DCM, −20° C. to rt; E) 9, pTsOH, dioxane, 100° C., 15 min.

Step-1

A mixture of 499 mg of 1 (2.19 mmol), 547 mg of 2 (2.19 mmol), and 500uL of N,N-diisopropylethylamine in 20 mL of anhydrous tetrahydrofuranwas heated at 70° C. overnight. After cooling down, the reaction mixturewas concentrated, and subject to aqueous workup with 50 mL of EtOAc, 20mL of sodium bicarbonate solution, brine, and dried over anhydroussodium sulfate. After filtration and concentration, the residue waspassed through a short silica cartridge, eluted with heptanes/EtOAc (v/v3/1), giving 815 mg of a slight yellow solid (84%). LC-MS: m/z 441.0(ES+), 439.0 (ES−).

Step-2

A mixture of intermediate 3 (815 mg, 1.85 mmol), 4 (740 mg, 1.1 equiv.),27 mg of dichlorobis(triphenylphosphine)palladium (II) (2% mol) in 6 mLof anhydrous DMF was purged with nitrogen for 30 min. The reactionmixture was then heated at 70° C. overnight. LC-MS showed 70%conversion. After cooling down, 30 mL of ethyl acetate and 760 mg ofpotassium fluoride in 5 mL of water was added, and the mixture wasstirred at rt for at least 2 hr. The white precipitate was filtered out,and the organic layer was separated, washed with water, brine, and driedover anhydrous sodium sulfate.

After filtration and concentration, the residue was dissolved in 20 mLof acetone, followed by addition of 3 mL of 1.0 N aqueous HCl solution.The mixture was heated at 60° C. for 15 min, and concentrated underreduced pressure. Normal workup was done using 50 mL of EtOAc, 10 mL ofsaturated sodium bicarbonate solution, brine, anhydrous sodium sulfate.After concentration, the residue was purified by flash columnchromatography on silica gel, giving 405 mg of yellow solid (70% basedon consumed starting material), also recovering intermediate 3 183 mg.LC-MS: m/z 405.1 (ES+), 403.1 (ES−).

Step-3

To a mixture of 1.28 g of intermediate I-2 in 10 mL of dichloromethane,was added 10 mL of 4.0 N HCl in dioxane. After stirring at rt overnight,the solvent was removed, and the residue was dried in vacuum. LC-MS: m/z305.1 (ES+), 303.1 (ES−).

Step-4

Under N₂, to a mixture of the intermediate 6 obtained above, 1 mL ofDIPEA in 10 mL of NMP and 10 mL of dichloromethane at −20° C., was added275 uL of 7 (1.1 equiv). The reaction was continued for 5 min, thenquenched with 1 mL of isopropyl alcohol. The reaction mixture was warmedup to rt, and extracted with 100 mL of EtOAc, washed with water 10 mL×2,brine, dried over sodium sulfate. After filtration and concentration,the residue was purified by flash column chromatography with eluentheptanes/EtOAc (v/v 2/3), giving yellow solid I-3 450 mg (40%). LC-MS:m/z 359.1 (ES+), 357.1 (ES−).

Step-5

The mixture of 30 mg intermediate 8 (84 μmol) and 13 mg of 9 (1.2 equiv)in 1 mL of 0.08 M p-TsOH dioxane solution was heated at 100° C. for 15min. After cooling down, the reaction mixture was subject to regularwork up with 50 mL of EtOAc, aqueous sodium bicarbonate, brine, anddried over anhydrous sodium sulfate. After concentration, the residuewas purified by column chromatography on silica gel with heptane/EtOAc(v/v 1/4) as eluent, giving 22.8 mg pale white solid (61%). LC-MS:m/z=447.1 (ES+), 445.2 (ES−).

Example 43 Preparation of1-{6-[5-Acetyl-2-(4-morpholin-4-yl-phenylamino)-pyrimidin-4-ylamino]-2,3-dihydro-benzo[1,4]oxazin-4-yl}-propenoneI-169

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using4-morpholin-4-yl-phenylamine in the place of 9 in Step 5. LC-MS: m/z501.1 (ES+), 499.2 (ES−).

Example 44 Preparation of1-{6-[5-Acetyl-2-(6-morpholin-4-yl-pyridin-3-ylamino)-pyrimidin-4-ylamino]-2,3-dihydro-benzo[1,4]oxazin-4-yl}-propenoneI-168

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using3-amino-[6-morpholin-4-yl]-pyridine in the place of 9 in Step 5. LC-MS:m/z 502.2 (ES+), 500.3 (ES−).

Example 45 Preparation of1-{6-[5-Acetyl-2-(1-methyl-1H-indazol-6-ylamino)-pyrimidin-4-ylamino]-2,3-dihydro-benzo[1,4]oxazin-4-yl}-propenoneI-154

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using1-methyl-1H-indazol-6-ylamine in the place of 9 in Step 5. LC-MS: m/z470.1 (ES+), 468.1 (ES−).

Example 46 Preparation of1-{6-[5-Acetyl-2-(1H-indazol-6-ylamino)-pyrimidin-4-ylamino]-2,3-dihydro-benzo[1,4]oxazin-4-yl}-propenoneI-153

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using 1H-indazole-6-ylamine inthe place of 9 in Step 5. LC-MS: m/z 456.1 (ES+), 454.2 (ES−).

Example 47 Preparation of1-{4-[5-Acetyl-4-(4-acryloyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-ylamino)-pyrimidin-2-ylamino]-phenyl)}-pyrrolidin-2-oneI-152

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using1-(4-amino-phenyl)-pyrrolidin-2-one in the place of 9 in Step 5. LC-MS:m/z 456.1 (ES+), 454.2 (ES−).

Example 48 Preparation of1-(6-{5-Acetyl-2-[4-(2-methoxy-ethoxy)-phenylamino]-pyrimidin-4-ylamino}-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propenoneI-150

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using4-(2-methoxy-ethoxy)-phenylamine in the place of 9 in Step 5. LC-MS: m/z490.2 (ES+), 488.3 (ES−).

Example 49 Preparation of5-[5-Acetyl-4-(4-acryloyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-ylamino)-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-oneI-129

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using5-amino-1,3-dihydro-indol-2-one in the place of 9 in Step 5. LC-MS: m/z471.1 (ES+), 469.2 (ES−).

Example 50 Preparation of1-(6-{5-Acetyl-2-[6-(2-hydroxy-ethoxy)-pyridin-3-ylamino]-pyrimidin-4-ylamino}-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propenoneI-128

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using2-(5-amino-pyridin-2-yloxy)-ethanol in the place of 9 in Step 5. LC-MS:m/z 477.1 (ES+), 475.2 (ES−).

Example 51 Preparation ofN-{3-[5-Acetyl-2-(6-methoxy-pyridin-3-ylamino)-pyrimidin-4-ylamino]-phenyl}-acrylamideI-189

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using tert-butyl3-aminophenylcarbamate in the place of 2 in Step 1 and5-amino-2-methoxypyridine in the place of 9 in Step 5. LC-MS: m/z 405.1(ES+), 403.2 (ES−).

Example 52 Preparation ofN-{3-[5-Acetyl-2-(6-methoxy-pyridin-3-ylamino)-pyrimidin-4-yloxy]-phenyl}-acrylamideI-188

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using tert-butyl3-hydroxyphenylcarbamate in the place of 2 in Step 1 and5-amino-2-methoxypyridine in the place of 9 in Step 5. LC-MS: m/z 406.2(ES+), 404.1 (ES−).

Example 53 Preparation of1-{3-[5-Acetyl-2-(6-methoxy-pyridin-3-ylamino)-pyrimidin-4-ylamino]-azetidin-1-yl}-propenoneI-187

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using 3-amino-N-Boc-azetidinein the place of 2 in Step 1 and 5-amino-2-methoxypyridine in the placeof 9 in Step 5. LC-MS: m/z 369.1 (ES+), 367.2 (ES−).

Example 54 Preparation ofN-(3-{5-Acetyl-2-[4-(2-methoxy-ethoxy)-phenylamino]-pyrimidin-4-ylamino}-phenyl)-acrylamideI-124

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using tert-butyl3-aminophenylcarbamate in the place of 2 in Step 1 and4-(2-methoxy-ethoxy)-phenylamine in the place of 9 in Step 5. LC-MS: m/z448.2 (ES+), 446.3 (ES−).

Example 55 Preparation ofN-(3-{5-Acetyl-2-[6-(2-methoxy-ethoxy)-pyridin-3-ylamino]-pyrimidin-4-ylamino}-phenyl)-acrylamideI-122

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using tert-butyl3-aminophenylcarbamate in the place of 2 in Step 1 and6-(2-Methoxy-ethoxy)-pyridin-3-ylamine in the place of 9 in Step 5.LC-MS: m/z 449.2 (ES+), 447.1 (ES−).

Example 56 Preparation ofN-(3-{5-Acetyl-2-[6-(2-hydroxy-ethoxy)-pyridin-3-ylamino]-pyrimidin-4-ylamino}-phenyl)-acrylamideI-121

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by using tert-butyl3-aminophenylcarbamate in the place of 2 in Step 1 and2-(5-Amino-pyridin-2-yloxy)-ethanol in the place of 9 in Step 5. LC-MS:m/z 435.1 (ES+), 433.2 (ES−).

Example 57 Preparation of4-(3-acrylamidophenoxy)-2-(3-methoxyphenylamino)-pyrimidine-5-carboxylicacid phenylamide I-200

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, NaH, THF, 0° C.; B) NaOH, THF, MeOH; C) 5, TBTU, DIPEA, CH₃CN, 0°C.; D) MCPBA, CH₂Cl₂, 0° C.; E) 8, 50° C., 3 h; F) TFA, CH₂Cl₂; G) 11,DIPEA, CH₂Cl₂

Step 1

To a stirred solution of (3-hydroxyphenyl)carbamic acid tert-butyl ester2 (1.79 g, 8.59 mmol) at 0° C. was added a suspension of sodium hydride(60% dispersion in mineral oil) (0.34 g, 8.9 mmol) in anhdyrous THF (30mL). The mixture was stirred at 0° C. for 20 minutes. The phenoxidesolution was then added dropwise at 0° C. to a solution of4-chloro-(2-methylsulfanyl)pyrimidine-5-carboxylic acid ethyl ester 1 (2g, 8.59 mmol) in THF (20 mL). The reaction mixture was stirred at 0° C.for 2 hours. The reaction mixture was diluted with ethyl acetate (150mL) and washed with water (50 mL) and then brine (50 mL). The organiclayer was dried over sodium sulphate, filtered and concentrated invacuo. The crude product was washed with CH₂Cl₂:hexane (1:9) to affordthe title compound 3 as a white solid (2.43 g, 70%).

Step-2

To a stirred solution of4-(3-tert-butoxycarbonylaminophenoxy)-2-(methylsulfanyl-pyrimidine)-5-carboxylicacid ethyl ester 3 (2 g, 4.93 mmol) in THF (60 mL), was added methanol(60 mL) at −10° C., followed by aqueous sodium hydroxide (0.3 g, 30 mLwater, 7.5 mmol). The reaction mixture was allowed warm to roomtemperature and was stirred for 1 hour. The reaction mixture was dilutedwith water (50 mL), acidified with citric acid and the resulting solidwas collected by filtration and washed with ice cold water (50 mL) toyield 4 as a white solid. (1.52 g, 82%).

Step-3

To a stirred solution of4-(3-tert-butoxycarbonylaminophenoxy)-2-(methylsulfanyl)pyrimidine-5-carboxylicacid 4 (2.0 g, 5.29 mmol) and TBTU (2.55 g, 7.94 mmol) in acetonitrile(30 mL) at 0° C. was added DIPEA (1.36 g, 10.6 mmol) followed by aniline5 (0.60 g, 6.35 mmol). The reaction was stirred at room temperature for2 hours. After completion of the reaction the reaction mixture waspoured into ice cold water (100 mL) and the white solid obtained wascollected by filtration and washed with ice cold water (20 mL), driedunder in vacuo to afford the title compound 6 (1.79 g, 75%).

Step-4

To a stirred solution of[3-(2-methylsulfanyl-5-phenylcarbamoylpyrimidin-4-yloxy)-phenyl]-carbamicacid tert-butyl ester 6 (1.5 g, 3.31 mmol) in CH₂Cl₂ at 0° C. was addeda solution m-CPBA (70%, 1.62 g, 2 eq) in CH₂Cl₂ (10 mL). The reactionmixture was allowed to warm to room temperature and was stirred for 12h. The reaction was quenched with saturated aqueous NaHCO₃ and the wholewas extracted with EtOAc. The organic layer was washed with brine, driedover Na₂SO₄, filtered, and concentrated in vacuo. The crude product waswashed with CH₂Cl₂:hexane (1:9) to afford the title compound 7 as awhite solid (1.16 g, 73%).

Step-5

Excess 3-methoxyaniline (8) (2 mL) was added to solid[3-(2-methanesulfonyl-5-phenylcarbamoyl-pyrimidin-4-yloxy)-phenyl]-carbamicacid tert-butyl ester 7 (0.5 g, 1.03 mmol) and the resulting mixture washeated to 50° C. under an argon atmosphere for 3 hours. The reactionmixture was cooled to room temperature and diluted with ethylacetate/hexane (1:1, 20 mL) and the resulting precipitate filtered andwashed with ethyl acetate/hexane (1:1, 10 mLl) to afford the desiredproduct 9 as a white solid (0.40 g, 75% yield).

Step-6

To a solution of{3-[2-(3-methoxy-phenylamino)-5-phenylcarbamoyl-pyrimidin-4-yloxy]-phenyl}-carbamicacid tert-butyl ester 9 (0.3 g, 0.56 mmol) in CH₂Cl₂ (10 mL) was addedtrifluoroacetic acid (2 mL) and the mixture was stirred at roomtemperature for 1 hour. Solvents were removed under reduced pressure andthe residue was dissolved in CH₂Cl₂, washed with 10% aqueous NaHCO₃solution, dried (Na₂SO₄), filtered, and evaporated under reducedpressure to provide the free amine 10 as white solid.

Step-7

To a stirred solution of amine 10 (0.24 g, 0.56 mmol) in dichloromethane(20 mL) under argon atmosphere cooled to −70° C. was added DIPEA (0.072g, 0.56 mmol) followed by drop wise addition of acryloyl chloride (0.050g, 0.56 mmol). The resulting mixture was stirred at −70° C. for 5minutes, and the reaction mixture diluted with CH₂Cl₂ (50 mL) and thenwas washed with saturated aqueous NaCl solution (10 mL). The organiclayer was dried (Na₂SO₄), filtered and evaporated under reducedpressure. The residue was purified by flash chromatography on silica gelusing (MeOH—CHCl₃ 5:95) as eluent to provide the target compound 11(0.094 g, 35%) as white solid: ¹H NMR (200 MHz, DMF-d7) δ 8.9 (s, 1H),8.10-7.70 (m, 6H), 7.60-7.10 (m, 6H), 6.60 (m, 2H) 6.40 (dd, 1H, J=8.0,2.0 Hz), 5.80 (m, 2H), 3.70 (s, 3H).

Example 58 Preparation of4-(3-acrylamidophenoxy)-2-(6-methoxypyridin-3-ylamino)-pyrimidine-5-carboxylicacid phenylamide I-159

The title compound was prepared according to the schemes, steps andintermediates described in Example 57 by using 6-methoxy-3-aminopyridinein place of 8 in Step 5. ¹H NMR (200 MHz, DMSO-d₆ δ 8.90 (s, 1H), 8.20(brs, 1H), 7.90-7.60 (m, 4H), 7.45 (m, 4H), 7.10 (m, 2H), 6.50 (m, 1H),6.20 (m, 2H), 5.90 (dd, J=8.0, 2.0 Hz, 1H), 3.90 (s, 3H).

Example 59 Preparation of4-(3-acrylamidophenoxy)-2-(3-methoxyphenylamino)-pyrimidine-5-carboxylicacid cyclopropylamide I-177

The title compound was prepared according to the schemes, steps andintermediates described in Example 57 by using cyclopropylamine in placeof 5 in Step 3. ¹H NMR (200 MHz, CD₃OD) δ 9.0 (s, 1H), 7.90 (brs, 1H),7.50 (m, 3H), 7.0 (m, 4H), 6.50 (m, 1H), 6.40 (d, J=8.0 Hz, 2H), 5.80(dd, J=8.2, 3.0 Hz, 1H), 3.60 (s, 3H), 0.90 (m, 2H), 0.62 (m, 2H).

Example 60 Preparation of4-(3-acrylamidophenoxy)-2-(6-methoxypyridin-3-ylamino)-pyrimidine-5-carboxylicacid cyclopropylamide I-176

The title compound was prepared according to the schemes, steps andintermediates described in Example 57 by using cyclopropylamine in placeof 5 in Step 3 and 6-methoxy-3-aminopyridine in place of 8 in Step 5. ¹HNMR (200 MHz, CD₃OD) δ 8.90 (s, 1H), 7.95 (brs, 1H), 7.90-7.82 (m, 3H),7.40 (m, 3H), 6.98 (d, J=6.0 Hz, 1H), 6.42 (m, 2H), 5.90 (dd, J=8.0, 2.0Hz, 1H), 3.90 (s, 3H), 0.95 (m, 2H), 0.83 (m, 2H).

Example 61 Preparation of4-(3-acrylamidophenoxy)-2-(3-methoxyphenylamino)pyrimidine-5-carboxylicacid amide I-178

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, NaH, THF, 0° C.; B) LiOH, THF, H₂O; C) 5, TBTU, DIPEA, CH₃CN; D)MCPBA, CHCl₃, 0° C.; E) 8, DMA, 90° C., 24 h; F) 4N HCl, dioxane; G) 11,CH₂Cl₂; H) triflic acid, TFA, CH₂Cl₂

Step-1

Step 1 was carried out in a manner similar to Step 1 in Example 57.

Step-2

Saponification of 3 (4.58 g, 11.3 mmol) by LiOH (500 mg, 20 mmol) in 80mL THF/H2O (1:1) and usual workup with 1 N HCl gave free acid 4.

Step-3

Acid 4 was directly mixed with 4-methoxybenzylamine (1.55 g, 11.3 mmol),TBTU (5.4 g, 16.8 mmol) and DIEA (2.4 mL, 13.4 mmol) in 100 mL MeCN atroom temperature. The reaction mixture was run overnight to give 6 as awhite solid (4.2 g, 8.5 mmol) after flash chromatography (EtOAc-hexane).

Step-4

Step 4 was run in a manner similar to Step 4 in Example 57 with CHCl₃being substituted for CH₂Cl₂ as the solvent.

Step-5

The 2-methylsulfone of 7 (1.0 g, 1.9 mmol) was mixed with3-methoxyaniline (420 mg, 3.4 mmol) in DMA and the mixture was heated at90° C. for 24 hours. Workup was done in a manner similar to that forStep-5 in Example 57 to give 9 (300 mg, 0.52 mmol).

Steps-6, 7, and 8

The Boc group was removed from 9 by treatment with 4 N HCl in dioxane.The product (300 mg, 0.52 mmol) was treated immediately with acryloylchloride (43 μL, 0.52 mmol) in 15 mL DCM at −40° C. This intermediatewas purified by flash chromatography (MeOH-DCM) and was reacted withtriflic acid and TFA in DCM to provide the crude benzylamine 11 (120 mg,0.228 mmol). This intermediate (120 mg, 0.228 mmol) was converted toI-178 using triflic acid (305 μL, 3.44 mmol) in TFA/DCM (5 mL, 1:1) atroom temperature to provide ˜35 mg final compound I-178 as grey powderafter purification via column chromatography (16% yield for threesteps). MS: m/z=405.

Example 62 Preparation of tert-butyl3-(3-(4-(3-acrylamidophenylamino)-5-methylpyrimidin-2-ylamino)phenoxy)propylcarbamateI-45

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 1, methanesulfonyl chloride, CH₂Cl₂, Et3N, rt, 1 h; B) 3, K₂CO₃, DMF,60° C.; C) H₂, Pd/C, EtOH, rt, 16 hr; D) 6, 7, Pd(OAc)₂, BINAP, Cs₂CO₃,toluene, 100° C., 16 hr; E) 5, AcOH, EtOH, 90° C., 16 hr; F) H₂, Pd/C,EtOH, rt, 16 hr; G) 11, NMP, 0° C., 15 min

Step-1

To a stirring solution of 1 (1.0 g, 5.7 mmol) in dichloromethane (20.0mL) was added Et₃N (1.15 g, 11.41 mmol) and methanesulfonyl chloride(0.98 g, 8.56 mmol). The reaction mixture was stirred under nitrogenatmosphere at rt for 60 min. It was quenched with water (20 mL) andextracted with EtOAc (2×50 mL). The combined EtOAc extract was washedwith 10% NaHCO₃ soln. (25 mL), water (25 mL), brine (25 mL), dried overNa₂SO₄ and concentrated under reduced pressure to get 2 (1.36 g, 94%) asa colorless viscous liquid. It was used in the next step without furtherpurification.

Step-2

To a stirring solution of 2 (0.749 g, 5.39 mmol) and K₂CO₃ (0.99 g, 7.19mmol) in dry DMF (20 mL) was added 3 (1.36 g, 5.39 mmol) and thereaction mixture was heated at 60° C. for 16 h under nitrogenatmosphere. It was cooled, concentrated under reduced pressure and theresidue was taken in ethyl acetate (25 mL). The ethyl acetate soln. waswashed with water (2×10 mL), brine (10 mL), dried over Na₂SO₄ andconcentrated under reduced pressure to get 4 (1.2 g, 75%) as a yellowishviscous liquid. It was used in the next step without furtherpurification.

Step-3

To a solution of 4 (1.20 g, 4.05 mmol) in ethanol (25 mL)) was addedPd/C (0.12 g, 10% w/w) and the reaction mixture was allowed to stirunder H₂ atmosphere (1.5 Kg hydrogen pressure) at rt for 16 h. Thereaction mixture was filtered through a pad of Celite® and concentratedunder reduced pressure to get 5 (0.95 g, 88%) as a brownish viscous oil.It was used in the next step without further purification.

Step-4

To a solution of 6 (1.69 g, 12.26 mmol), in toluene (50.0 mL) was added7 (2.0 g, 12.26 mmol), BINAP (0.3 g, 0.49 mmol), cesium carbonate (7.9g, 24.5 mmol). The solution was degassed (by purging N₂ for 15 min) andto it was added Pd(OAc)₂ (0.054 g, 0.25 mmol). The reaction mixture wasstirred at 100° C. for 16 h under nitrogen atmosphere. It was cooled,diluted with ethyl acetate (100 mL) and filtered through Celite®. Thefiltrate was washed with water (2×25 mL), brine (25 mL), dried overNa₂SO₄ and concentrated under reduced pressure. The residue obtained wasfurther purified by column chromatography (SiO₂, 60-120 mesh,Ethylacetete/hexane: 15/85). The solid obtained after evaporating therequired fractions was washed with diethyl ether and dried under highvacuum to get 8 (1.2 g, 37%) as a light yellow solid.

Step-5

To a solution of 8 (0.5 g, 1.89 mmol) and 5 (0.805 g, 3.0 mmol) inethanol (10.0 mL) was added glacial acetic acid (0.056 g, 0.95 mmol),and the reaction mixture was stirred in a sealed tube for 16 h at 90° C.The reaction mixture was cooled, concentrated under reduced pressure.The residue was quenched with 10% sodium bicarbonate soln. (10.0 mL) andextracted with ethyl acetate (3×15 mL). The combined ethyl acetateextract was washed with water (15 mL), brine (15 mL), dried over Na₂SO₄and concentrated under reduced pressure to get a residue. The cruderesidue was further purified by column chromatography (SiO₂,EtOAc/Hexane: 50/50) to get 9 (0.57 g, 61%) as a light yellow solid.

Step-6

To a solution of 9 (0.56 g, 1.13 mmol) in ethanol (25 mL)) was added 10%Pd/C (0.068 g) and the reaction mixture was allowed to stir under H₂atmosphere (1.5 Kg hydrogen pressure) at rt for 16 h. The reactionmixture was filtered through a pad of Celite® and concentrated underreduced pressure to get 10 (0.45 g, 85%) as a brownish solid. It wasused in the next step without further purification.

Step-7

To a stirred solution of 10 (0.25 g, 0.5382 mmol) in NMP (2.5 mL) at 0°C. was added acryloyl chloride (0.073 g, 0.807 mmol) and the reactionmixture was stirred at 0° C. for 15 min The reaction mixture was addeddrop wise to a cold, stirring solution of 10% NaHCO₃. After completeaddition the solution was stirred for another 30 min at 0° C., and thenfiltered through a Buchner funnel to isolate the precipitated solid. Thesolid was washed with cold water and hexane. It was dissolved inmethanol:dichloromethane (50:50, 10 mL) and concentrated under reducedpressure. The residue obtained was suspended in cold water (50 mL), Et₃Nwas added to it and it was extracted with ethyl acetate (2×100 mL). Thecombined ethyl acetate extract was washed with water (50 mL), brine (50mL), dried over Na₂SO₄ and concentrated under reduced pressure to getI-45 (0.100 g, 35.8%) as an off-white solid. ¹H NMR (DMSO-d₆) δ ppm:1.37 (s, 9H), 1.70-1.80 (m, 2H), 2.10 (s, 3H), 3.00-3.06 (m, 2H), 3.79(t, J=6.24 Hz, 2H), 5.74 (d, J=11.92 Hz, 1H), 6.24 (dd, J=1.84 & 15.16Hz, 1H), 6.35-6.47 (m, 2H), 6.80-6.90 (bs, 1H), 6.97 (t, J=8.28 Hz, 1H),7.23-7.27 (m, 2H), 7.31 (s, 1H), 7.37 (d, J=8.2 Hz, 1H), 7.46 (d, J=7.48Hz, 1H), 7.90-7.90-7.91 (m, 2H), 8.36 (s, 1H), 8.87 (s, 1H), 10.07 (s,1H); LCMS: m/e 519 (M+1).

Example 63 Preparation of tert-butyl3-(3-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)phenoxy)propylcarbamateI-183

The title compound was prepared according to the schemes, steps andintermediates described below.

A) methanesulfonyl chloride, CH₂Cl₂, Et₃N, rt, 1 h; B) K₂CO₃, DMF, 60°C., 16 h; C) Pd—C, H₂, ethanol, rt, 16 h.

Step 1′

To a stirred solution of 2′ (4.0 g, 22.8 mmol) in dichloromethane (80.0mL) was added Et₃N (4.6 g, 45.5 mmol) and methanesulfonyl chloride (3.92g, 34.2 mmol), and the reaction mixture was stirred under nitrogenatmosphere at RT for 60 min. The reaction was quenched with water (50mL) and extracted with EtOAc (2×100 mL). The combined extracts werewashed with 10% NaHCO₃ solution (50 mL), water (50 mL), and brine (50mL), dried over Na₂SO₄, and concentrated under reduced pressure to give2 (5.5 g, 95.2%) as a light yellow viscous liquid. Compound 2 was usedin the next step without further purification.

Step 2′

To a stirred solution of 1 (2.3 g, 16.5 mmol) and K₂CO₃ (4.6 g, 33.3mmol) in dry DMF (100 mL) was added 2 (5.5 g, 21.7 mmol), and thereaction mixture was heated at 60° C. for 16 h under nitrogenatmosphere. The reaction was cooled, quenched with water (250 ml), andextracted with EtOAc (2×100 mL). The combined extracts were washed with10% NaHCO₃ solution (100 mL), water (3×100 mL), and brine (100 mL),dried over Na₂SO₄, and concentrated under reduced pressure to give 3(4.0 g, 81.6%) as a light yellow viscous liquid. Compound 3 was used inthe next step without further purification.

Step 3′

To a solution of 3 (4.0 g, 13.4 mmol) in ethanol (50 mL) was added Pd/C(0.8 g, 10% w/w), and the reaction mixture was allowed to stir under H₂atmosphere (1.5 Kg hydrogen pressure) at rt for 16 h. The reactionmixture was filtered through a pad of Celite® and concentrated underreduced pressure to give 4 (3.3 g, 91.9%) as a brownish viscous oil.Compound 4 was used in the next step without further purification.

Step 1

A pressure tube was charged with 2 (10.0 g, 0.072 mol), 1 (24.1 g, 0.145mol), n-BuOH (100 mL) and DIPEA (13.9 g, 0.108 mol), and the contentswere stirred at 120° C. for 2 h. The reaction mixture was cooled, andthe precipitated solid was isolated by filtration through a Buchnerfunnel, washed with cold hexane and dried to give 3 (12.5 g, 64%) as ayellow solid. Compound 3 was used in the next step without furtherpurifications.

Step 2

To a solution of 3 (1.5 g, 5.58 mmol) and 4 (1.48 g, 5.58 mmol) inethanol (30.0 mL) was added glacial acetic acid (0.167 g, 2.79 mmol),and the reaction mixture was stirred in a pressure tube at 90° C. for 48h. The reaction mixture was cooled and concentrated under reducedpressure; the residue was quenched with 10% sodium bicarbonate solution(20.0 mL) and extracted with ethyl acetate (2×50 mL). The combinedextracts were washed with water (25 mL) and brine (25 mL), dried overNa₂SO₄, and concentrated under reduced pressure to give crude 5. Thecrude residue was purified by column chromatography (neutral Al₂O₃,MeOH/Chloroform: 0.5/99.5) to give 5 (1.4 g, 50.3%) as a brown solid.

Step 3

To a solution of 5 (1.4 g, 2.8 mmol) in ethanol (50 mL)) was added 10%Pd/C (0.28 g, 10% w/w) and the reaction mixture was allowed to stirunder H₂ atmosphere (1.5 Kg hydrogen pressure) at rt for 16 h. Thereaction mixture was filtered through a pad of Celite® and concentratedunder reduced pressure to give a residue. The crude residue was furtherpurified by column chromatography (neutral Al₂O₃, MeOH/Chloroform:0.5/99.5) to give a solid which was washed with dichloromethane/hexanemixtures to give 6 (0.7 g, 53.4%) as a pale brown solid.

Step 4

tert-Butyl3-(3-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)phenoxy)propylcarbamate

To a stirred solution of 6 (0.25 g, 0.533 mmol) and potassium carbonate(0.138 g, 1.02 mmol) in NMP (2.5 mL) at 0° C. was added acryloylchloride (0.060 g, 0.665 mmol), and the reaction mixture was stirred at0° C. for 30 min. The reaction mixture was added dropwise to a cold,stirring solution of 10% NaHCO₃ and stirred at the same temperature (0°C.) for 30 min. A white solid precipitated out and was isolated byfiltration through a Buchner funnel. The solid was washed with coldwater and hexane and dissolved in mixture of methanol/dichloromethane(50:50, 10 mL) and concentrated under reduced pressure. The residueobtained was suspended in cold water (25 mL), Et₃N was added, and it wasextracted with ethyl acetate (2×50 mL). The combined extracts werewashed with water (50 mL), brine (50 mL), dried over Na₂SO₄ andconcentrated under reduced pressure to give I-183 (0.255 g, 91.4%) aslight yellow solid. ¹H NMR (DMSO-d₆) δ ppm: 1.36 (s, 9H), 1.78 (quin,J=6.4 Hz, 2H), 3.01-3.06 (m, 2H), 3.83 (t, J=6.12 Hz, 2H), 5.74 (dd,J=1.4 & 10.04 Hz, 1H), 6.24 (d, J=16.84 Hz, 1H), 6.41-6.48 (m, 2H), 6.88(s, 1H), 7.03 (t, J=8.24 Hz, 1H), 7.23-7.31 (m, 3H), 7.41 (d, J=8.28 Hz,1H), 7.56 (d, J=7.96 Hz, 1H), 7.90 (s, 1H), 8.11 (d, J=3.56 Hz, 1H),9.11 (s, 1H), 9.43 (s, 1H), 10.10 (s, 1H); LCMS: m/e 523.1 (M+1).

Example 64 Preparation of tert-butyl3-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)phenoxy)propylcarbamateI-198

The title compound was prepared according to the schemes, steps andintermediates described in Example 63 by using tert-butyl3-(4aminophenoxy)propylcarbamate in place of 4 in Step-2. ¹H NMR(DMSO-d₆) δ ppm: 1.37 (s, 9H), 1.78 (quin, J=6.36 Hz, 2H), 3.05 (q,J=6.24 Hz, 2H), 3.86 (t, J=6.2 Hz, 2H), 5.75 (dd, J=1.92 & 10.04 Hz,1H), 6.24 (dd, J=1.92 & 16.92 Hz, 1H), 6.45 (dd, J=10.08 & 16.92 Hz,1H), 6.72 (d, J=9 Hz, 2H), 6.89 (t, J=5.4 Hz, 1H), 7.26 (t, J=8.08 Hz,1H), 7.40 (d, J=8.12 Hz, 1H), 7.48-7.52 (m, 3H), 7.92 (s, 1H), 8.05 (d,J=3.72 Hz, 1H), 8.95 (s, 1H), 9.36 (s, 1H), 10.12 (s, 1H); LCMS: m/e523.2 (M+1).

The intermediate tert-butyl 3-(4aminophenoxy)propylcarbamate wasprepared by the scheme shown below.

A) NaH, THF, rt, 16 h; B) H₂, Pd/C, EtOH, rt, 16 hr

Step-1

To a stirring solution of 1 (1.7 g, 9.7 mmol) in dry THF (40 mL) wasadded NaH (0.72 g, 18.0 mmol, 60% dispersion in paraffin oil) at 0° C.and the reaction mixture was stirred at rt for 15 min under nitrogenatmosphere. To it was added 2 (2.0 g, 13.87 mmol) and the reactionmixture was stirred at rt for 16 h. It was quenched with cold water (20mL), and extracted with ethyl acetate (25 mL). The ethyl acetate extractwas washed with water (2×10 mL), brine (10 mL), dried over Na₂SO₄ andconcentrated under reduced pressure to get an oily liquid which wastriturated with hexane to get 3 (2.0 g, 69.5%) as a yellow crystallinesolid.

Step-2

To a solution of 3 (2.0 g, 6.749 mmol) in ethanol (30 mL)) was added 10%Pd/C (0.4 g, 20% w/w) and the reaction mixture was allowed to stir underH₂ atmosphere (1.5 Kg hydrogen pressure) at rt for 16 h. The reactionmixture was filtered through a pad of Celite® and concentrated underreduced pressure to get 4 (1.6 g, 89.3%) as a pinkish viscous oil. Itwas used in the next step without further purification.

Example 65 Preparation of 4-(3acrylamidophenylamino)-5-fluoro-2-(3,4-dimethoxyphenylamino)-pyrimidineI-134

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using 3,4-dimethoxyaniline inplace of 4 in Step-2. ¹H NMR (200 MHz, CD₃OD) δ 8.50 (s, 1H), 7.80 (d,J=6.5 Hz, 1H), 7.70-7.66 (m, 2H), 7.20 (m, 1H), 7.0 (m, 2H), 6.41 (m,2H), 5.92 (dd, J=8.0, 2.0 Hz, 1H), 3.89 (s, 6H).

Example 66 Preparation of4-(3-acrylamidophenylamino)-5-fluoro-2-(3,4,5-trimethoxyphenylamino)-pyrimidineI-133

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using 3,4,5-trimethoxyanilinein place of 4 in Step-2. ¹H NMR (200 MHz, CD₃OD) δ 8.10 (s, 1H), 8.0 (d,J=6.0 Hz, 1H), 7.50 (m, 2H), 7.30 (m, 1H), 7.0 (m, 2H), 6.45 (m, 2H),5.90 (dd, J=8.0, 2.0 Hz, 1H), 3.90 (s, 3H), 3.89 (s, 9H).

Example 67 Preparation of4-(3-acrylamidophenylamino)-5-fluoro-2-(3-(hydroxymethyl)phenylamino)-pyrimidineI-145

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using 3-hydroxymethylaniline inplace of 4 in Step-2. ¹H NMR (DMSO-d₆) δ ppm: 4.38 (d, J=5.6 Hz, 2H),5.07 (t, J=5.68 Hz, 1H), 5.75 (d, J=10.84 Hz, 1H), 6.24 (dd, J=16.96 Hz,1H), 6.44 (dt, J=10.04 & 17.0 Hz, 1H), 6.83 (d, J=7.4 Hz, 1H), 7.10 (t,J=7.72 Hz, 1H), 7.28 (t, J=8.16 Hz, 1H), 7.40 (d, J=8.08 Hz, 1H),7.55-7.59 (m, 3H), 7.92 (s, 1H), 8.09 (d, J=3.6 Hz, 1H), 9.11 (s, 1H),9.40 (s, 1H), 10.1 (s, 1H); LCMS: m/e 378.0 (M+1).

Example 68 Preparation of4-(3-acrylamidophenylamino)-5-fluoro-2-(3-(3-(2-oxopyrrolidin-1-yl)propoxy)phenylamino)-pyrimidineI-144

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-(2-oxopyrrolidin-1-yl)propoxyaniline in place of 4 in Step-2. ¹H NMR(DMSO-d₆) δ ppm: 1.8-1.94 (m, 4H), 2.18 (q, J=8.08 Hz, 2H), 3.26-3.40(m, 4H), 3.80 (t, J=6 Hz, 2H), 5.74 (d, J=10.72 Hz, 1H), 6.24 (d,J=15.64 Hz, 1H), 6.41-6.80 (m, 2H), 7.04 (t, J=8.16 Hz, 1H), 7.22-7.29(m, 2H), 7.33 (s, 1H), 7.42 (d, J=8.08 Hz, 1H), 7.55 (d, J=7.52 Hz, 1H),7.91 (s, 1H), 8.11 (d, J=3.48 Hz, 1H), 9.13 (s, 1H), 9.43 (s, 1H), 10.11(s, 1H); LCMS: m/e 491 (M+1).

Example 69 Preparation of4-(3-acrylamidophenylamino)-5-fluoro-2-(3-(3-(methylsulfonyl)propoxy)phenylamino)-pyrimidine I-138

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-(3-(methylsulfonyl)propoxyaniline in place of 4 in Step-2. ¹H NMR(DMSO-d₆) δ ppm: 2.05-2.15 (m, 2H), 3.0 (s, 3H), 3.22 (t, J=7.76 Hz,2H), 3.93 (t, J=6.08 Hz, 2H), 5.74 (dd, J=1.88 & 10 Hz, 1H), 6.25 (dd,J=1.8 & 16.88 Hz, 1H), 6.44 (dd, J=10.16 & 16.84 Hz, 2H), 7.05 (t,J=8.16 Hz, 1H), 7.24-7.30 (m, 2H), 7.35 (s, 1H), 7.42 (d, J=8.2 Hz, 1H),7.55 (d, J=8 Hz, 1H), 7.90 (s, 1H), 8.11 (d, J=3.6 Hz, 1H), 9.14 (s,1H), 9.43 (s, 1H), 10.10 (s, 1H); LCMS: m/e 484 (M+1).

The intermediate 3-(3-(methylsulfonyl)propoxyaniline was prepared by thescheme shown below.

A) DEAD, Ph₃P, Et₃N, THF, rt, 1 hr; B) MCPBA, CH₂Cl₂, rt, 30 min; C)TFA, CH₂Cl₂, rt, 1 hr

Step-1

To a stirred solution of 2 (1.1 g, 10.3 mmol) in THF (20 mL) were added1 (2.18 g, 10.3 mmol), PPh₃ (2.98 g, 11.3 mmol) and Et₃N (1.68 g, 15mmol) under N₂ atmosphere. The reaction mixture was cooled to 0° C. andto it was added DEAD (1.98 g, 11.3 mmol). The reaction mixture wasallowed to come to rt and stirred for 1 h. It was quenched with water,extracted with ethyl aceate (3×25 mL) and the combined EtOAc extract waswashed with water and brine solution (5 mL each). The residue obtainedafter concentration under reduced pressure was purified by columnchromatography (SiO₂, 60-120, pet ether/ethyl acetate, 8/2) to get 3 (2g 60.6%) as a white solid.

Step-2

To a stirred solution of 3 (2 g, 6.7 mmol) in CH₂Cl₂ (25 mL) was addedm-CPBA (4.13 g, 26.7 mmol) at −10° C. The reaction mixture was allowedto come to rt and stirred for 30 min. It was quenched with Na₂CO₃solution (10 mL), extracted with CH₂Cl₂ (10 mL), dried over Na₂SO₄,filtered and concentrated under reduced pressure. The residue wasfurther purified by column chromatography (SiO₂, 60-120,chloroform/methanol 9/1) to get 4 (1.05 g, 68.8%) as a yellow oil.

Step-3

To a stirred solution of 4 (0.75 g, 2.2 mmol) in CH₂Cl₂ (7.5 mL) wasadded TFA (3 vol.) at 0° C. The reaction mixture was allowed to come tort and stirred further at it for 1 h. It was concentrated under reducedpressure, basified with NaHCO₃ solution (5 mL) and extracted with CH₂Cl₂(3×10 mL). The combined organic extract was washed with water (2 mL) andbrine solution (2 mL). Drying over Na₂SO₄ followed by filtration andconcentration under reduced pressure offered 5 (500 mg, 96%) as brownsolid.

Example 70 Preparation ofN-(3-(5-fluoro-2-(3-(2-hydroxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-105

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-(2-hydroxy)ethoxyaniline in place of 4 in Step-2. ¹H NMR (DMSO-d₆) δppm: 3.67 (dd, J=4.5 & 10 Hz, 2H), 3.85-3.87 (m, 2H), 4.83 (t, J=5.6 Hz,1H), 5.75 (bd, J=10 Hz, 1H), 6.25 (d, J=15.6 Hz, 1H), 6.42-6.46 (m, 2H),7.05 (t, J=8.4 Hz, 1H), 7.26 (d, J=8 Hz, 1H), 7.30 (d, J=8 Hz, 1H), 7.33(s, 1H), 7.41 (d, J=8 Hz, 1H), 7.57 (d, J=8 Hz, 1H), 7.92 (s, 1H), 8.11(d, J=3.6 Hz, 1H), 9.11 (s, 1H), 9.42 (s, 1H), 10.11 (s, 1H); LCMS: m/e409.9 (M+1).

The intermediate 3-(2-hydroxy)ethoxyaniline was prepared by the schemeshown below.

A) K₂CO₃, DMF, 70° C., 12 h; B) Pd—C, H₂, ethanol, rt, 10 h; C) 1M LAHsolution, THF, −15° C., 45 min.

Step-1

To a stirring solution of 1 (2.0 g, 14.37 mmol) and K₂CO₃ (3.95 g, 28.6mmol) in dry DMF (15 mL) was added 2 (2.88 g, 17.25 mmol) and thereaction was stirred at rt 70° C. for 12 h under nitrogen atmosphere.The reaction mixture was cooled, concentrated under reduced pressure andthe residue was diluted with ethyl acetate (50 mL). It was washed withwater (2×10 mL), brine (10 mL), dried over Na₂SO₄ and concentrated underreduced pressure to get 3 (2.5 g, 78%) as a light brown liquid. It wasused in the next step without further purification.

Step-2

To a solution of 3 (2.0 g, 8.88 mmol) in ethanol (20 mL)) was added Pd/C(0.2 g, 10% w/w) and the reaction mixture was allowed to stir under H₂atmosphere (1.0 Kg hydrogen pressure) at rt for 10 h. The reactionmixture was filtered through a pad of Celite® and concentrated underreduced pressure to get 4 (1.6 g, 94%) as a light brown liquid. It wasused in the next step without further purification.

Step-3

To a stirring solution of 4 (1.2 g, 6.14 mmol) in dry THF (12 mL)) wasadded lithium aluminum hydride (9.2 mL, 9.20 mmol, 1.0 M soln. in THF)at −15° C., under N₂ atmosphere. The reaction mixture was allowed tocome to rt and stirred at it for 45 min. The reaction mixture wasquenched with saturated ammonium chloride solution and was filteredthrough a pad of Celite® and extracted with EtOAc (2×20 mL). Thecombined organic layer was washed with brine (10 mL) and concentratedunder reduced pressure to get A (0.9 g, 95%) as a dark brown liquid. Itwas used in the next step without further purification.

Example 71 Preparation ofN-(3-(5-fluoro-2-(3-(2-hydroxy-2-methylpropoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-118

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 110° C., 16 h; B) Pd(OAc)₂, BINAP, Cs₂CO₃, toluene,100° C., 16 h; C) MeMgBr (3M solution in ether), THF, −78° C., 3 h; D)TFA, CH₂Cl₂, rt, 3 h. E) K₂CO₃, NMP, rt, 45 min.

Step-1

Compound 3 was prepared according to the schemes, steps andintermediates described in Example 20.

Step-2

A solution of 4 (0.7 g, 3.5 mmol), 3 (1.45 g, 4.3 mmol), Pd(OAc)₂ (0.03g, 0.14 mmol), BINAP (0.13 g, 0.21 mmol) and Cs₂CO₃ (2.8 g, 8.7 mmol) indegassed toluene (30 mL) (toluene was purged with N₂ for 30 min) washeated at 100° C. for 16 h under N₂ atmosphere. The reaction mixture wascooled, diluted with EtOAc (15 mL) and washed with water (10 mL), brine(10 mL) and dried over Na₂SO₄. Filtration followed by concentrationunder reduced pressure offered a residue which was further purified bycolumn chromatography (SiO₂, 60-120, pet ether/ethyl acetate, 6/4) toget 5 (700 mg, 40%) as a white solid.

Step-3

To a stirred solution of 5 (0.4 g, 0.8 mmol) in THF (10 mL) was addedMethyl magnesium bromide ((3 M solution in ether, 1.6 mL, 4.8 mmol) at−78° C. The reaction mixture was allowed to warm to −30° C. over 3 h,cooled again to −78° C. and quenched with saturated ammonium chloridesolution (5 mL). The mixture was filtered through Celite® and filtratewas concentrated under reduced pressure to afford 6 as a pale yellowsolid (300 mg, 78%) which was taken for next step without furtherpurification.

Step-4

To a stirred solution of 6 (0.2 g, 0.4 mmol) in CH₂Cl₂ (7.5 mL) wasadded TFA (3 vol.) at 0° C. The reaction mixture was allowed to come tort and stirred further at it for 3 h. It was concentrated under reducedpressure, basified with NaHCO₃ solution (5 mL) and extracted with CH₂Cl₂(3×10 mL). The combined organic extract was washed with water (2 mL) andbrine solution (2 mL). Drying over Na₂SO₄ followed by filtration andconcentration under reduced pressure afforded a residue which wasfurther purified by column chromatography (SiO₂, 60-120, pet ether/ethylacetate, 6/4) to get 7 (130 mg, 86%) as a white solid.

Step-5

To a stirred solution of 7 (0.08 g, 0.2 mmol) and potassium carbonate(0.11 g, 0.8 mmol) in NMP (1 mL) at 0° C. was added 8 (0.023 g, 0.22mmol) and the reaction mixture was stirred at 0° C. for 45 min Thereaction mixture was added drop wise to a cold, stirring solution of 10%NaHCO₃ and stirred at the same temperature (0° C.) for 30 min. A solidprecipitated out which was isolated by filtration through a Buchnerfunnel. The solid was washed with cold water, hexane and dissolved in amixture of methanol/dichloromethane (50:50, 5 mL) and concentrated underreduced pressure. The residue obtained was suspended in cold water (10mL), Et₃N was added to it and it was extracted with ethyl acetate (2×5mL). The combined ethyl acetate extract was washed with water (2 mL),brine (2 mL), dried over Na₂SO₄ and concentrated under reduced pressure.The residue obtained was further purified by column chromatography(SiO₂, 60-120, pet ether/ethyl acetate, 5/5) to get I-118 (35 mg, 38%)as a white solid. ¹H NMR (CD₃OD) δ ppm: 1.27 (s, 6H), 3.67 (s, 2H), 5.76(dd, J=2.4 & 9.6 Hz, 1H), 6.34 (dd, J=2 & 16.8 Hz, 1H), 6.42 (dd, J=9.6& 16.8 Hz, 1H), 6.54 (td, J=2 & 7.2 Hz, 1H), 7.07-7.12 (m, 2H),7.27-7.31 (m, 2H), 7.40 (d, J=8 Hz, 1H), 7.45 (d, J=8 Hz, 1H), 7.92 (d,J=4 Hz, 1H), 8.07 (d, J=2 Hz, 1H); LCMS: m/e 436.2 (M−1).

Example 72 Preparation ofN-(3-(5-fluoro-2-(3-(2-morpholino-2-oxoethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-110

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using4-[(3-aminophenoxy)acetyl]-morpholine in place of 4 in Step-2. ¹H NMR(DMSO-d₆) δ ppm: 3.4-3.5 (bm, 4H), 3.5-3.6 (bm, 4H), 4.69 (s, 2H), 5.75(dd, J=2 & 10 Hz, 1H), 6.25 (dd, J=2 & 17.2 Hz, 1H), 6.42-6.49 (m, 2H),7.05 (t, J=8 Hz, 1H), 7.29 (t, J=8 Hz, 3H), 7.41 (d, J=8 Hz, 1H), 7.57(d, J=8.8 Hz, 1H), 7.91 (s, 1H), 8.12 (d, J=3.6 Hz, 1H), 9.15 (s, 1H),9.45 (s, 1H), 10.12 (s, 1H); LCMS: m/e 491.0 (M−2).

The intermediate 4-[(3-aminophenoxy)acetyl]-morpholine was prepared bythe scheme shown below.

A) LiOH, THF, MeOH, H₂O, rt, 4 h; B) SOCl₂, 85° C., morpholine, 0° C.,30 min; C) Pd—C, H₂, ethyl acetate, rt, 2 h.

Step-1

To a stirred solution of 1 (1.0 g, 4.44 mmol) in methanol/THF/water: 5mL/5 mL/5 mL was added LiOH monohydrate (0.75 g, 17.76 mmol) and thereaction mixture was stirred at rt for 4 h. It was concentrated underreduced pressure, the residue was diluted with water (10 mL), acidifiedwith 1.0 N HCl (PH˜5-6) and extracted with ether (2×20 mL). The combinedether extract was washed with water (20 mL), brine (20 mL), dried overNa₂SO₄ and concentrated under reduced pressure to get 2 (0.8 g, 91.43%)as an off-white solid.

Step-2

Thionyl chloride (2.0 ml, 27.56 mmol) was added to 2 (0.2 g, 1.014 mmol)under nitrogen atmosphere. A drop of N,N Dimethylformamide was added tothe mixture and the contents were stirred at 85° C. for 2 h. Aftercooling to rt thionyl chloride was removed by concentration underreduced pressure. The residue was cooled to 0° C., morpholine (0.5 g,5.74 mmol) was added to it in small portions and the reaction mixturewas stirred at 0° C. for 30 min. The reaction mixture was allowed tocome to rt and stirred at it for 30 min, cooled and quenched with water(10 mL). The contents were extracted with ether (2×10 mL) and thecombined ether extract was washed with water (5 mL), brine (5 mL), driedover Na₂SO₄ and concentrated under reduced pressure to get 3 (0.180 g,66.67%) as a yellow solid.

Step-3

To a solution of 3 (0.180 g, 0.676 mmol) in ethyl acetate (10 mL)) wasadded Pd/C (0.036 g, 20% w/w) and the reaction mixture was allowed tostir under H₂ atmosphere (1.0 Kg hydrogen pressure) at rt for 2 h. Thereaction mixture was filtered through a pad of Celite® and concentratedunder reduced pressure to get A (0.14 g, 87.67%) as an off-white solid.It was used in the next step without further purifications.

Example 73 Preparation ofN-(3-(5-fluoro-2-(3-(1-hydroxy-2-methylpropan-2-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-91

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-(1-hydroxy-2-methylpropan-2-yloxy)aniline in place of 4 in Step-2. ¹HNMR (DMSO-d₆) δ ppm: 1.16 (s, 6H), 3.32-3.35 (m, 2H), 4.81 (t, J=5.74Hz, 1H), 5.74 (dd, J=1.84 & 10.04 Hz, 1H), 6.24 (dd, J=1.88 & 16.96 Hz,1H), 6.44 (dd, J=10.12 & 16.96 Hz, 1H), 6.50 (dd, J=2.12 & 7.96 Hz, 1H),7.02 (t, J=8.12 Hz, 1H), 7.26-7.30 (m, 2H), 7.41 (d, J=8.16 Hz, 1H),7.48 (d, J=8.24 Hz, 1H), 7.57 (d, J=8.12 Hz, 1H), 7.92 (s, 1H), 8.09 (d,J=3.6 Hz, 1H), 9.07 (s, 1H), 9.41 (s, 1H), 10.09 (s, 1H); LCMS: m/e438.0 (M+1).

The intermediate 3-(1-hydroxy-2-methylpropan-2-yloxy)aniline wasprepared by the scheme shown below.

A) K₂CO₃, DMF, 16 h, rt; B) Pd/C, ethanol, 5 h, rt; C) LAH (1M in THFsolution), 0° C. to rt, 2 h.

Step-1

To a solution of 1 (0.5 g 3.59 mmol) and 2 (0.84 g 4.316 mmol) in DMFwas added K₂CO₃ (0.99 g, 7.194 mmol). After stirring at rt for 16 h,reaction mixture was concentrated under reduced pressure. The residuewas diluted with ethyl acetate (10 mL) and washed with 10% NaOH solution(5 mL), water (5 mL) and brine solution (5 mL). Drying over Na₂SO₄,followed by concentration under reduced pressure gave 3 as red brownliquid (0.5 g, 52%).

Step-2

To a stirred solution of 3 (0.45 g, 1.77 mmol) in ethanol (5 mL) wasadded Pd/C (45 mg) and the reaction mixture was hydrogenated (bladderpressure, ˜1.5 Kg) for 5 h. The reaction mixture was passed through aCelite® bed and concentrated under vacuum to get 4 (0.35 g, 88%) as acolorless liquid.

Step-3

To a stirred solution of 4 (0.25 g, 1.15 mmol) in THE (5 mL) under N₂was added LAH (3.45 mL, 3.35 mmol, 1M solution in THF) at 0° C. Thereaction mixture was allowed to come to rt and stirred at it for 2 h. Itwas carefully quenched with saturated Na₂SO₄ solution (2 mL), filteredand concentrated. The residue was further purified by columnchromatography (SiO₂, 60-120, pet ether/ethyl acetate, 6/4) to give 5 asa light brown liquid (0.15 g, 71%).

Example 74 Preparation ofN-(3-(5-fluoro-2-(3-(2-(2-oxopyrrolidin-1-yl)ethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-164

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-(2-(2-oxopyrrolidin-1-yl)ethoxy)aniline in place of 4 in Step-2. ¹HNMR (DMSO-d₆) δ ppm: 1.89 (quin, J=7.6 Hz, 2H), 2.21 (t, J=8 Hz, 2H),3.40 (t, J=6.8 Hz, 2H), 3.50 (t, J=5.6 Hz, 2H), 3.93 (t, J=5.2 Hz, 2H),5.75 (dd, J=2 & 10 Hz, 1H), 6.25 (dd, J=2 & 16.84 Hz, 1H), 6.42-6.49 (m,2H), 7.05 (t, J=8.4 Hz, 1H), 7.28 (t, J=8 Hz, 2H), 7.33 (s, 1H), 7.43(d, J=8 Hz, 1H), 7.57 (d, J=8 Hz, 1H), 7.92 (s, 1H), 8.12 (d, J=3.6 Hz,1H), 9.15 (s, 1H), 9.45 (s, 1H), 10.13 (s, 1H); LCMS: m/e 475 (M−2).

Example 75 Preparation ofN-(3-(5-fluoro-2-(6-(3-(methylsulfonyl)propoxy)pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-80

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-amino-6-(3-(methylsulfonyl)propoxy)pyridine in place of 4 in Step-2.¹H NMR (DMSO-d₆) δ ppm: 2.05-2.20 (m, 2H), 3.00 (s, 3H), 3.24 (t, J=7.46Hz, 2H), 4.27 (t, J=6.32 Hz, 2H), 5.75 (dd, J=1.76 & 10 Hz, 1H), 6.25(dd, J=1.8 & 16.96 Hz, 1H), 6.45 (dd, J=10.04 & 16.92 Hz, 1H), 6.65 (d,J=8.88 Hz, 1H), 7.27 (t, J=8.08 Hz, 1H), 7.39 (d, J=8.08 Hz, 1H), 7.49(d, J=8 Hz, 1H), 7.92 (s, 1H), 7.99 (dd, J=2.6 & 8.76 Hz, 1H), 8.07 (d,J=3.64 Hz, 1H), 8.31 (d, J=2.28 Hz, 1H), 9.10 (s, 1H), 10.11 (s, 1H);LCMS: m/e 486.9 (M+1).

Example 76 Preparation ofN-(3-(2-(6-cyclobutoxypyridin-3-ylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-79

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-amino-6-cyclobutoxypyridine in place of 4 in Step-2. ¹H NMR (DMSO-d₆)δ ppm: 1.57-1.66 (m, 1H), 1.71-1.78 (m, 1H), 1.94-2.04 (m, 2H),2.32-2.38 (m, 2H), 4.95-5.05 (m, 1H), 5.73-5.76 (m, 1H), 6.25 (dd,J=1.92 & 16.92 Hz, 1H), 6.45 (dd, J=10.08 & 16.92 Hz, 1H), 6.58 (d,J=8.84 Hz, 1H), 7.25 (t, J=8.04 Hz, 1H), 7.39 (d, J=7.84 Hz, 1H),7.45-7.55 (m, 1H), 7.90 (s, 1H), 7.94 (dd, J=2.72 & 8.88 Hz, 1H), 8.06(d, J=3.68 Hz, 1H), 8.27 (d, J=2.6 Hz, 1H), 9.04 (s, 1H), 9.41 (s, 1H),10.1 (s, 1H); LCMS: m/e 421.2 (M+1).

Example 77 Preparation ofN-(3-(5-fluoro-2-(6-((1-methylpiperidin-4-yl)methoxy)pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-78

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-amino-6-(1-methylpiperidin-4-yl)methoxypyridine in place of 4 inStep-2. ¹H NMR (DMSO-d₆) δ ppm: 1.23-1.27 (m, 3H), 1.65-1.69 (m, 2H),1.83 (t, J=11.72 Hz, 2H), 2.14 (s, 3H), 2.75 (d, J=11.24 Hz, 2H), 4.0(d, J=6.2 Hz, 2H), 5.74 (dd, J=2 & 10.04 Hz, 1H), 6.24 (dd, J=1.96 &16.92 Hz, 1H), 6.45 (dd, J=10.08 & 16.92 Hz, 1H), 6.62 (d, J=8.88 Hz,1H), 7.27 (t, J=8.08 Hz, 1H), 7.40 (d, J=8.88 Hz, 1H), 7.47 (d, J=7.6Hz, 1H), 7.92 (s, 1H), 7.97 (dd, J=2.76 & 8.92 Hz, 1H), 8.07 (d, J=3.72Hz, 1H), 8.28 (d, J=2.64 Hz, 1H), 9.07 (s, 1H), 9.41 (s, 1H), 10.11 (s,1H); LCMS: m/e 478.0 (M+1).

Example 77 Preparation ofN-(3-(5-fluoro-2-(4-chloro-3-methoxyphenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-74

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using 4-chloro-3-methoxyanilinein place of 4 in Step-2. ¹H NMR (DMSO-d₆) δ ppm: 3.64 (s, 3H), 5.74 (dd,J=2.12 & 9.96 Hz, 1H), 6.24 (dd, J=1.84 & 17 Hz, 1H), 6.44 (dd, J=10 &16.84 Hz, 1H), 7.13 (s, 1H), 7.28 (t, J=8.08 Hz, 1H), 7.36 (dd, J=2.12 &8.68 Hz, 1H), 7.40 (d, J=7.64 Hz, 1H), 7.45 (d, J=2.04 Hz, 1H), 7.50 (d,J=8.12 Hz, 1H), 7.91 (s, 1H), 8.13 (d, J=3.52 Hz, 1H), 9.28 (s, 1H),9.48 (s, 1H), 10.12 (s, 1H); LCMS: m/e 414.0 (M+1).

Example 78 Preparation ofN-(3-(5-fluoro-2-(4-(2-hydroxy-2-methylpropoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-73

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using4-(2-hydroxy-2-methylpropoxy)aniline in place of 4 in Step-2. ¹H NMR(MeOD) δ ppm: 1.33 (s, 6H), 3.75 (s, 2H), 5.80 (dd, J=3.28 & 10.64 Hz,1H), 6.39 (dd, J=2.24 & 16.96 Hz, 1H), 6.47 (dd, J=9.6 & 16.96 Hz, 1H),6.84 (td, J=3.48 & 9.0 Hz, 2H), 7.30 (t, J=7.72 Hz, 1H), 7.41-7.50 (m,4H), 7.89 (d, J=3.88 Hz, 1H), 8.09 (s, 1H); LCMS: m/e 438 (M+1).

Example 79 Preparation ofN-(3-(5-fluoro-2-(6-(1,1-dioxidothiomorpholin-4-yl)pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamide I-72

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-amino-6-(1,1-dioxidothiomorpholin-4-yl) pyridine in place of 4 inStep-2. ¹H NMR (DMSO-d₆) δ ppm: 3.00-3.15 (bm, 4H), 3.90-4.10 (bm, 4H),5.76 (dd, J=1.64 & 10.04 Hz, 1H), 6.26 (dd, J=1.72 & 16.92 Hz, 1H), 6.46(dd, J=10.04 & 16.88 Hz, 1H), 6.87 (d, J=9.04 Hz, 1H), 7.20 (t, J=8.04Hz, 1H), 7.39 (d, J=8.24 Hz, 1H), 7.50 (d, J=7.68 Hz, 1H), 7.90-7.93 (m,2H), 8.06 (d, J=3.6 Hz, 1H), 8.35 (d, J=2.4 Hz, 1H), 9.0 (s, 1H), 9.40(s, 1H), 10.12 (s, 1H); LCMS: m/e 484 (M+1).

Example 80 Preparation ofN-(3-(5-fluoro-2-(6-(2-(2-oxopyrrolidin-1-yl)ethoxy)pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-70

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-amino-6-(2-(2-oxopyrrolidin-1-yl)ethoxy)pyridine in place of 4 inStep-2. ¹H NMR (DMSO-d₆) δ ppm: 1.90 (quintet, J=7.6 Hz, 2H), 2.19 (t,J=8.04 Hz, 2H), 3.41 (t, J=6.88 Hz, 2H), 3.50 (t, J=5.36 Hz, 2H), 4.27(t, J=5.48 Hz, 2H), 5.75 (d, J=10.92 Hz, 1H), 6.25 (d, J=17.04 Hz, 1H),6.45 (dd, J=10.12 & 16.84 Hz, 1H), 6.63 (d, J=8.96 Hz, 1H), 7.27 (t,J=8.04 Hz, 1H), 7.39 (d, J=7.56 Hz, 1H), 7.47 (d, J=7.32 Hz, 1H), 7.92(s, 1H), 7.98 (dd, J=2.36 & 8.84 Hz, 1H), 8.08 (d, J=3.3 Hz, 1H), 8.31(d, J=2.24 Hz, 1H), 9.10 (s, 1H), 9.44 (s, 1H), 10.11 (s, 1H); LCMS: m/e478.0 (M+1).

Example 81 Preparation of(R)—N-(3-(5-fluoro-2-(6-(tetrahydrofuran-3-yloxy)pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-69

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using(R)-3-amino-6-(tetrahydrofuran-3-yloxy)pyridine in place of 4 in Step-2.¹H NMR (DMSO-d₆) δ ppm: 1.91-1.99 (m, 1H), 2.14-2.23 (m, 1H), 3.70-3.77(m, 2H), 3.81 (dd, J=7.90 & 15.48 Hz, 1H), 3.88 (dd, J=4.76 & 10.16 Hz,1H), 5.38 (t, J=4.68 Hz, 1H), 5.75 (dd, J=1.72 & 10.08 Hz, 1H), 6.24 (d,J=16.92 Hz, 1H), 6.45 (dd, J=10.16 & 16.88 Hz, 1H), 6.63 (d, J=8.84 Hz,1H), 7.26 (d, J=7.64 Hz, 1H), 7.39 (d, J-=7.92 Hz, 1H), 7.46 (d, J=7.64Hz, 1H), 7.92 (s, 1H), 7.97 (dd, J=2.6 & 8.83 Hz, 1H), 8.07 (d, J=3.6Hz, 1H), 8.32 (d, J=2.48 Hz, 1H), 9.08 (s, 1H), 9.42 (s, 1H), 10.10 (s,1H); LCMS: m/e 437.2 (M+1).

Example 82 Preparation ofN-(3-(2-(4-chloro-3-(3-(methylsulfonyl)propoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-55

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using4-chloro-3-(3-(methylsulfonyl)propoxy)aniline in place of 4 in Step-2.¹H NMR (DMSO-d₆) δ ppm: 2.07-2.14 (m, 2H), 3.0 (s, 3H), 3.22 (t, J=7.72Hz, 2H), 3.90 (t, J=6.08 Hz, 2H), 5.75 (dd, J=1.88 & 10.08 Hz, 1H), 6.24(dd, J=1.84 & 16.92 Hz, 1H), 6.44 (dd, J=10.12 & 16.96 Hz, 1H), 7.15 (d,J=8.72 Hz, 1H), 7.30 (t, J=8.08 Hz, 1H), 7.35 (dd, J=2.2 & 8.8 Hz, 1H),7.43 (d, J=8 Hz, 1H), 7.45-7.55 (m, 2H), 7.91 (s, 1H), 8.14 (d, J=3.56Hz, 1H), 9.31 (s, 1H), 9.49 (s, 1H), 10.14 (s, 1H); LCMS: m/e 520.0(M+1).

Example 83 Preparation ofN-(3-(2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-96

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in Step-2. ¹H NMR(DMSO, 400 MHz) δ 10.13 (s, 1H), 9.43 (s, 1H), 9.18 (s, 1H), 8.09 (d,1H, J=3.68 Hz), 7.92 (s, 1H), 7.65 (dd, 1H, J=2.3, 14.2 Hz), 7.47 (d,1H, J=8.24 Hz), 7.41 (d, 1H, J=8.28 Hz), 7.27 (t, 2H, J=8.0 Hz), 6.94(t, 1H, J=9.4 Hz), 6.44 (dd, 1H, J=16.96, 10.1 Hz), 6.23 (dd, 1H,J=1.84, 16.96 Hz), 5.73 (dd, 1H, J=1.4, 10.1 Hz), 4.04 (m, 2H), 3.61 (m,2H), 3.29 (s, 3H). MS m/z: 442.0 (M+H⁺).

Example 84 Preparation ofN-(3-(2-(4-tert-butoxycarbonyl-2,3-dihydrobenzo[1,4]oxazin-6-yl)amino-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-175

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using6-amino-4-tert-butoxycarbonyl-2,3-dihydrobenzo[1,4]oxaxine in place of 4in Step-2. MS m/z: 507.1 (M+H⁺).

Example 85 Preparation ofN-(3-(2-(4-tert-butoxycarbonyl-2,3-dihydrobenzo[1,4]oxazin-6-yl)amino-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-174

The title compound was prepared by treating the product of Example 84with 4N HCl in dioxane at rt for 1 hr followed by removal of solvents invacuo. MS m/z: 407.1 (M+H⁺).

Example 86 Preparation ofN-(3-(2-(4-trifluoroacetyl-2,3-dihydrobenzo[1,4]oxazin-6-yl)amino-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-143

The title compound was prepared by treating the product of Example 85with trifluoroacetic anhydride at rt for 1 hr followed by removal ofsolvents in vacuo. MS m/z: 503.1 (M+H⁺).

Example 87 Preparation ofN-(3-(2-(4-methylsulfonyl-2,3-dihydrobenzo[1,4]oxazin-6-yl)amino-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-140

The title compound was prepared by treating the product of Example 85with mesyl chloride Et₃N in CH₂Cl₂ at 0° C. for 30 min, followed bywashing with aqueous NaHCO₃, drying over Na₂SO₄ and removal of solventsin vacuo. MS m/z: 485.1 (M+H⁺).

Example 88 Preparation ofN-(3-(2-(4-methyl-2,3-dihydrobenzo[1,4]oxazin-6-yl)amino-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-126

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using6-amino-4-methyl-2,3-dihydrobenzo[1,4]oxazine in place of 4 in Step-2.MS m/z: 421.1 (M+H⁺).

Example 89 Preparation ofN-(3-(2-(4-acetyl-2,3-dihydrobenzo[1,4]oxazin-6-yl)amino-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-112

The title compound was prepared by treating the product of Example 85with acetic anhydride and pyridine in CH₂Cl₂ at rt for 1 hr, followed bywashing with 1N HCl, then with aqueous NaHCO₃, drying over Na₂SO₄ andremoval of solvents in vacuo. MS m/z: 449.1 (M+H

Example 90 Preparation ofN-(3-(2-(1-tert-butoxycarbonyl-1H-indazol-5-yl)amino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-151

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using5-amino-N-(tert-butoxycarbonyl)-1H-indazole in place of 4 in Step-2. MSm/z: 490.2 (M+H⁺).

Example 91 Preparation ofN-(3-(2-(1H-indazol-5-yl)amino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-156

The title compound was prepared by treating the product of Example 90with 4N HCl in dioxane at rt for 1 hr followed by removal of solvents invacuo. MS m/z: 390.1 (M+H⁺).

Example 92 Preparation ofN-(3-(2-(1-methyl-1H-indazol-5-yl)amino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-155

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using5-amino-1-methyl-1H-indazole in place of 4 in Step-2. MS m/z: 404.2(M+H⁺).

Example 93 Preparation ofN-(3-(5-fluoro-2-(3-sulfamoylphenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-160

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-butanol, 120° C., 2 h, pressure tube; B) AcOH, ethanol, 90°C., 16 h; C) Pd—C, H₂, ethanol, rt, 3 h; D) acryloyl chloride, K₂CO₃,NMP, 0° C., 60 min.

Step-1

A pressure tube was charged with 2 (10.0 g, 0.072 mol), 1 (24.1 g, 0.145mol), n-BuOH (100 mL) and DIPEA (13.9 g, 0.108 mol) and the contentswere stirred at 120° C. for 2 h. The reaction mixture was cooled, theprecipitated solid was isolated by filtration through a Buchner funnel,washed with cold hexane and dried to get 3 (12.5 g, 64%) as a yellowsolid. It was used in the next step without further purification.

Step-2

To a solution of 3 (0.25 g, 0.93 mmol) and 4 (0.16 g, 0.93 mmol) inethanol (2.5 mL) was added glacial acetic acid (0.083 g, 1.39 mmol), andthe reaction mixture was stirred in a pressure tube at 90° C. for 16 h.It was cooled, the precipitated solid was isolated by filtration througha Buchner funnel, washed with cold ether and dried to get 5 (0.245 g,65%) as brown solid. It was used in the next step without furtherpurification.

Step-3

To a solution of 5 (0.1 g, 0.24 mmol in methanol (4 mL)) was added 10%Pd/C (0.2 g, 20% w/w) and the reaction mixture was allowed to stir underH₂ atmosphere (1.5 Kg hydrogen pressure) at rt for 3 h. The reactionmixture was filtered through a pad of Celite® and concentrated underreduced pressure to get 6 (0.076 g, 82%) as a brown solid. It was usedin the next step without further purification.

Step-4

To a stirred solution of 6 (0.07 g, 0.18 mmol) and potassium carbonate(0.051 g, 0.37 mmol) in NMP (0.7 mL) at 0° C. was added acryloylchloride (0.021 g, 0.23 mmol) and the reaction mixture was stirred at 0°C. for 60 min The reaction mixture was added drop wise to a cold,stirring solution of 10% NaHCO₃ and kept at the same temperature (0° C.)for 30 min. A solid precipitated out which was isolated by filtrationthrough a Buchner funnel. The solid was washed with cold water andhexane and dissolved in mixture of methanol/dichloromethane (50:50, 5mL) and concentrated under reduced pressure. The residue obtained wassuspended in cold water (10 mL), Et₃N was added to it and it wasextracted with ethyl acetate (2×10 mL). The combined ethyl acetateextract was washed with water (5 mL), brine (5 mL), dried over Na₂SO₄and concentrated under reduced pressure get a residue. The crude residuewas further purified by column chromatography (neutral Al₂O₃,MeOH/chloroform: 3/97) to get I-160 (0.028 g, 35%) as light brown solid.¹H NMR (DMSO-d₆) δ ppm: 5.75 (dd, J=1.68 & 10.24 Hz, 1H), 6.25 (dd,J=1.8 & 17 Hz, 1H), 6.43 (dd, J=10 & 16.92 Hz, 1H), 7.27-7.35 (m, 5H),7.40 (d, J=8 Hz, 1H), 7.60 (d, J=8.16 Hz, 1H), 7.92 (s, 1H), 7.95-8.05(m, 1H), 8.07 (s, 1H), 8.14 (d, J=3.52 Hz, 1H), 9.50 (s, 2H), 10.12 (s,1H); LCMS: m/e 428.9 (M+1).

Example 94 Preparation ofN-(3-(5-cyano-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-109

The title compound was prepared according to the steps and intermediatesas described below.

A) DMA, K₂CO₃, rt, 10 h, pressure tube; B) PTSA, dioxane, 100° C., 2 h;C) Zn(CN)₂, Ph₃P, DMF, 120° C., 12 h; D) 4N HCl, dioxane, rt, 1 hr; thenacryloyl chloride, Et₃N, DCM, −10° C., 10 min.

Step-1

To a solution of 5-bromo-2,4-dichloropyrimidine (0.45 g, 2.0 mmol) andtert-butyl 3-aminophenylcarbamate (0.44 g, 2.1 mmol) in DMA (3 mL) wasadded K₂CO₃ (0.55 g, 4.0 mmol). The suspension was stirred for 10 hours.Water (10 mL) was added and the precipitate was collected by filtration.The solid was washed with ether and dried to yield 0.8 g of compound 3.MS: m/e=399.1, 401.2 (M+1).

Step-2

To a solution of compound 3 (400 mg, 1.0 mmol) and4-(2-methoxyethoxy)aniline (0.2 g, 1.2 mmol) in 8 ml dioxane was added4-methylbenzenesulfonic acid monohydrate (0.15 g, 0.8 mmol). The mixturewas stirred at 100° C. for two hours. The solvent was evaporated. Theresidue was dissolved in 30 ml ethyl acetate and washed with NaHCO₃aqueous solution, water and brine. The organic layer was separated anddried over Na₂SO₄. After removal of solvent, the crude product wassubject to chromatography on silica gel (hexane:EtOAc=1:1). 0.40 g ofthe title compound 5 was obtained: MS m/z: 530.1, 532.1 (M+H⁺).

Step-3

To a suspension of Zn(CN)₂ (0.24 g, 2.0 mmol), Pd(PPh₃)₄ (60 mg, 0.05mmol) in 3 ml DMF was added to 5 (0.25 g, 0.5 mmol). The mixture wasdegassed and sealed under argon, and heated at 120° C. for 12 hours.Water (10 ml) was added and the precipitate was collected by filtration.The solid was washed with ether and dried to yield 0.2 g of compound 6.MS: m/e=477.1 (M+1).

Step-4

Compound 6 (0.10 g, 0.21 mmol) was dissolved in 4 N HCl (2 mL) indioxane. The mixture was stirred at rt for 1 hour. After removal ofsolvents, a 5-mL portion of DCM was poured in followed by evaporation todryness. This process of DCM addition followed by evaporation wasrepeated three times to give a residue solid which was used directly forthe next step: MS m/z: 377.0 (M+H⁺).

To a solution of the intermediate obtained above, triethylamine (0.1 ml,0.8 mmol) in 2 ml dichloromethane was added acryloyl chloride (19 mg,0.21 mmol) at −10° C. The reaction was stirred for 10 minutes at −10° C.and was quenched by NaHCO₃ aqueous solution. Ethyl acetate (10 mL) wasadded and washed with NaHCO₃ aqueous solution, water and brine. Theorganic layer was separated and dried over Na₂SO₄. After removal ofsolvent, the crude product was subject to chromatography on silica gel(hexane:EtOAc=1:2) to give 30 mg of the title compound. MS m/z: 431.1(M+H⁺).

Example 95 Preparation ofN-(3-(5-cyano-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-173

The title compound was prepared according to the schemes, steps andintermediates described in Example 94 by using 3-amino-6-methoxypyridinein place of 4 in Step-2. MS m/z: 388.2 (M+H⁺).

Example 96 Preparation ofN-(3-(5-cyclopropyl-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-139

The title compound was prepared according to the steps and intermediatesas described below.

A) Potassium cyclopropyltrifluoroborate, Pd(OAc)₂, Xanphos, Cs₂(CO₃),toluene, 100° C., 12 h; B) PTSA, dioxane, 100° C., 2 h; C) Zn(CN)₂,Ph₃P, DMF, 120° C., 12 h; D) 4N HCl, dioxane, rt, 1 hr; then acryloylchloride, Et₃N, DCM, −10° C., 10 min.

Step-1

Potassium cyclopropyltrifluoroborate (0.4 g, 3.0 mmol), compound 1 (1.0g, 2.5 mmol), palladium acetate (34 mg, 0.15 mmol), Xanphos (0.17 g, 0.3mmol) and Cs₂CO₃ (2.4 g, 7.5 mmol) were suspended in 25 ml toluene and 5ml water. The mixture was degassed, sealed under argon and heated at100° C. for 12 hours. 50 ml ethyl acetate was added and washed withNaHCO₃ aqueous solution, water and brine. The organic layer wasseparated and dried over Na₂SO₄. After removal of solvent, the crudeproduct was subject to chromatography on silica gel (hexane:EtOAc=3:2).0.54 g of the title compound 2 was obtained: MS m/z: 361.2 (M+H⁺).

Step-2

Compound 4 was prepared from compound 2 and 3 following the proceduredescribed in Step-2 of Example 94. MS m/z: 492.2 (M+H⁺).

Step-3

The title compound I-139 was prepared from compound 4 following theprocedure described in Step-4 of Example 94. MS m/z: 446.1 (M+H⁺).

Example 97 Preparation ofN-(3-(5-cyclopropyl-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-167

The title compound was prepared according to the schemes, steps andintermediates described in Example 96 by using 3-amino-6-methoxypyridinein place of 3 in Step-2. MS m/z: 403.2 (M+H⁺).

Example 98 Preparation ofN-(3-(5-fluoro-2-(3-(3-(2-oxopyrrolidin-1-yl)propoxy)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamideI-162

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIPEA, n-BuOH, 110° C., 16 h; B) Pd(OAc)₂, BINAP, Cs₂CO₃, toluene,100° C., 16 h; C) TFA, CH₂Cl₂, rt. 2 h; D) K₂CO₃, NMP, rt, 45 min.

Step-1

A pressure tube was charged with 2 (2.0 g, 9.61 mmol), 1 (3.21 g, 19.23mmol), n-BuOH (30 mL) and DIPEA (1.86 g, 14.42 mmol) and the contentswere stirred at 110° C. for 16 h. The reaction mixture was cooled,concentrated under reduced pressure, quenched with water (30 mL) andextracted with ethyl acetate (2×30 mL). The combined ethyl acetateextract was washed with water (20 mL), brine (20 mL), dried over Na₂SO₄and concentrated under reduced pressure to get a residue. It wastriturated with hexane to get 3 (2.5 g, 96%) as a yellow solid.

Step-2

To a solution of 3 (0.36 g, 1.1 mmol) in toluene (15 mL) was added3-(3-(2-oxopyrrolidin-1-yl)propoxyaniline 4 (0.25 g, 1.1 mmol) followedby BINAP (0.031 g, 0.05 mmol), palladium acetate (0.0022 g, 0.01 mmol),and Cs₂CO₃ (0.82 g, 2.5 mmol). The reaction mixture was stirred and N₂was bubbled into it for 15 min. It was heated at 100° C. for 8 h underN₂ atmosphere. The reaction mixture was cooled to room temperature,diluted with ethyl acetate (30 mL), washed with water (15 mL), brine (15mL), and dried over Na₂SO₄. Concentration under reduced pressure offereda residue which was purified by column chromatography (SiO₂, 60-120,product getting eluted in 3% methanol/chloroform: 3/97) to get 5 (0.3 g,60%) as yellow solid.

Step-3

To a stirred solution of 5 (0.25 g, 0.46 mmol) in CH₂Cl₂ (10 mL) wasadded TFA (1.0 mL) at 0° C. under nitrogen atmosphere. The reactionmixture was allowed to come to rt and stirred at this temperature for 2h. Crude reaction mixture was poured into ice cold water (10 mL),basified with sodium bicarbonate solution and extracted with ethylacetate (3×15 mL). The combined ethyl acetate extract was washed withwater (15 mL), brine (10 mL), dried over Na₂SO₄, and concentrated underreduced pressure to get 6 (0.130 g, 65%) as a yellow solid. It was usedin the next step without further purifications

Step-4

To a stirred solution of 6 (0.08 g, 0.18 mmol) and potassium carbonate(0.124 g, 0.9 mmol) in NMP (1.2 mL) at 0° C. was added acryloyl chloride(0.020 g, 0.22 mmol) and the reaction mixture was stirred at 0° C. for45 min The reaction mixture was added drop wise to a cold, stirringsolution of 10% NaHCO₃ and stirred at the same temperature (0° C.) for30 min. A solid precipitated out which was isolated by filtrationthrough a Buchner funnel. The solid was washed with cold water, hexaneand dissolved in a mixture of methanol/dichloromethane (50:50, 5 mL) andconcentrated under reduced pressure. The residue obtained was suspendedin cold water (10 mL), Et₃N was added to it and it was extracted withethyl acetate (2×10 mL). The combined ethyl acetate extract was washedwith water (5 mL), brine (5 mL), dried over Na₂SO₄ and concentratedunder reduced pressure to get I-162 (0.050 mg, 56%). ¹H NMR (DMSO-d₆) δppm: 1.81-1.91 (m, 4H), 2.19 (t, J=7.84 Hz, 2H), 3.26-3.35 (m, 4H), 3.73(t, J=6.04 Hz, 2H), 5.76 (dd, J=1.92 & 10.04 Hz, 1H), 6.25 (dd, J=1.88 &16.9 Hz, 1H), 6.38-6.45 (m, 2H), 6.93 (t, J=8.12 Hz, 1H), 7.02-7.04 (m,2H), 7.11 (s, 1H), 7.43 (t, J=8.16 Hz, 1H), 7.55 (d, J=8.24 Hz, 1H),7.68 (d, J=1.8 Hz, 1H), 8.56 (d, J=2.88 Hz, 1H), 9.56 (s, 1H), 10.34 (s,1H); LCMS: m/e 490.0 (M−2).

The intermediate 3-(3-(2-oxopyrrolidin-1-yl)propoxyaniline 4 wasprepared according to the scheme shown below.

A) NaH, DMF, rt, 16 h; B) SnCl₂, Conc. HCl, 50° C., 2 h.

Step-1

To a stirred solution of NaH (1.0 g, 20.94 mmol) in DMF (10 mL) wasadded 1 (2.0 g, 13.96 mmol) at 0° C. The reaction mixture was allowed tocome to rt and stirred at it for 30 mins. To the reaction mixture wasadded 2 (1.96 g, 13.96 mmol), slowly and the reaction mixture wasallowed to stir at rt for 16 h. The reaction mixture was concentratedunder reduced pressure and the residue was diluted with ethyl acetate(20 mL). It was washed with water (2×5 mL), brine (5 mL) and dried overNa₂SO₄. Filtration followed by concentration under reduced pressureoffered crude 3 (2 g, 55.5%) which was used in the next step withoutfurther purification.

Step-2

To a stirred solution of 3 (2 g, 7.57 mmol) in conc. HCl (20 mL) wasadded SnCl₂ (7.5 g, 34.06 mmol) in small portions. The reaction mixturewas stirred at 50° C. for 2 h, cooled and basified with NaHCO₃. It wasextracted with ethyl acetate (3×25 mL), washed with water (5 mL), brinesolution (5 mL) and dried over anhydrous Na₂SO₄. Filtration followed byconcentration under reduced pressure gave 4 (1.65 g, 93%) as dark brownsolid which was used as such in the next step.

Example 99 Preparation ofN-(4-(5-fluoro-2-(3-(2-(2-oxopyrrolidin-1-yl)ethoxy)phenylamino)pyrimidin-4-yloxy)benzyl)-N-methylacrylamideI-146

The title compound was prepared according to the schemes, steps andintermediates described in Example 98 by using(4-hydroxybenzyl)(methyl)carbamic acid tert-butyl ester in place of 2 inStep-1 and 3-(2-(2-oxopyrrolidin-1-yl)ethoxyaniline in place of 4 inStep-2. ¹H NMR (CDCl₃) δ ppm: 2.03 (quin, J=7.4 Hz, 2H), 2.39 (t, J=8Hz, 2H), 3.07 & 3.06 (s, together 3H), 3.57 (t, J=6.96 Hz, 2H), 3.66 (t,J=5.08 Hz, 2H), 4.03-4.04 (bd, J=4.96 Hz, 2H), 4.67 & 4.72 (s, together2H), 5.70-5.85 (m, 1H), 6.43 (d, J=16.72 Hz, 1H), 6.50 (d, J=5.72 Hz,1H), 6.60-6.75 (m, 1H), 6.89-6.96 (m, 2H), 7.06-7.08 (m, 2H), 7.18-7.30(m, 2H), 7.37 (d, J=8.44 Hz, 1H), 8.21 (d, J=2.36 Hz, 1H); LCMS: m/e506.2 (M+1).

Example 100 Preparation ofN-(4-(5-fluoro-2-(3-(3-(methylsulfonyl)propoxy)phenylamino)pyrimidin-4-yloxy)benzyl)-N-methylacrylamideI-136

The title compound was prepared according to the schemes, steps andintermediates described in Example 98 by using(4-hydroxybenzyl)(methyl)carbamic acid tert-butyl ester in place of 2 inStep-1 and 3-(3-(3-methylsulfonyl)propoxyaniline in place of 4 inStep-2. ¹H NMR (CDCl₃) δ ppm: 2.25-2.40 (m, 2H), 2.97 (s, 3H), 3.07 (s,3H), 3.20-3.30 (m, 2H), 3.98-4.05 (m, 2H), 4.67 (s, 1H), 4.72 (s, 1H),5.7-5.82 (m, 1H), 6.43 (dd, J=1.96 & 16.96 Hz, 1H), 6.49-6.53 (m, 1H),6.6-6.75 (m, 1H), 6.85-7.00 (m, 2H), 7.05-7.15 (m, 2H), 7.18-7.25 (m,2H), 7.36 (d, J=8.36 Hz, 1H), 8.21 (bd, J=2.52 Hz, 1H); LCMS: m/e 515.0(M+1).

Example 101 Preparation ofN-(4-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-yloxy)benzyl)-N-methylacrylamideI-117

The title compound was prepared according to the schemes, steps andintermediates described in Example 98 by using(4-hydroxybenzyl)(methyl)carbamic acid tert-butyl ester in place of 2 inStep-1 and 6-methoxy-3-aminopyridine in place of 4 in Step-2. ¹H NMR(DMSO-d₆) δ ppm: 2.92 & 3.07 (s, together 3H), 3.76 (s, 3H), 4.62 & 4.74(s, together 2H), 5.69 & 5.75 (dd, J=1.6 & 10.4 Hz, together 1H), 6.20(dd, J=1.2 & 16.4 Hz, 1H), 6.56 (d, J=8.8 Hz, 1H), 6.82-6.90 (m, 1H),7.28-7.35 (m, 4H), 7.71 (bd, J=7.6 Hz, 1H), 8.14 (s, 1H), 8.44 (bd,J=2.8 Hz, 1H), 9.48 (s, 1H); LCMS: m/e 410 (M+1).

Example 102 Preparation ofN-(4-(5-fluoro-2-(3-(3-(2-oxopyrrolidin-1-yl)propoxy)phenylamino)pyrimidin-4-yloxy)benzyl)-N-methylacrylamideI-111

The title compound was prepared according to the schemes, steps andintermediates described in Example 98 by using(4-hydroxybenzyl)(methyl)carbamic acid tert-butyl ester in place of 2 inStep-1 and 3-(3-(2-oxopyrrolidin-1-yl)propoxy)aniline in place of 4 inStep-2. ¹H NMR (DMSO-d₆) δ ppm: 1.80-2.6 (m, 4H), 2.20 (t, J=7.6 Hz,2H), 2.92 & 3.06 (s, together 3H), 3.20-3.40 (m, 4H), 3.75-3.90 (m, 2H),4.62 & 4.73 (s, together 2H), 5.65-5.77 (m, 1H), 6.20 (dd, J=2.4 & 16.8Hz, 1H), 6.24 (bd, J=8 Hz, 1H), 6.86 (dd, J=10.4 & 16.8 Hz, 1H), 6.93(t, J=8 Hz, 1H), 7.08 (t, J=8 Hz, 2H), 7.28-7.36 (m, 4H), 8.48 (d, J=2.8Hz, 1H), 9.51 (s, 1H); LCMS: m/e 520.2 (M+1).

Example 103 Preparation ofN-(3-(5-fluoro-2-(3-(2-(2-oxopyrrolidin-1-yl)ethoxy)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamideI-184

The title compound was prepared according to the schemes, steps andintermediates described below.

A)) K₂CO₃, DMF, rt, 16 h; B) Pd(OAc)₂ BINAP, Cs₂CO₃, toluene, 100° C., 8h; C) Pd—C, H₂, methanol, rt, 16 h. D)) acryloyl chloride, K₂CO₃, NMP,0° C., 30 min.

Step-1

To a stirring solution of 1 (24 g, 143.7 mmol) and K₂CO₃ (20 g, 143.6mmol) in dry DMF (300 mL) was added 2 (10 g, 71.8 mmol) and the reactionmixture was stirred at rt for 16 h under nitrogen atmosphere. It wascooled and quenched with water (600 mL). A white solid precipitated outwhich was isolated by filtration through Buchner funnel and vacuum driedto get 3 (13 g, 68%) as a white solid.

Step-2

To a solution of 3 (0.9 g, 3.3 mmol) in toluene (30 mL) was added 4 (950mg, 4.3 mmol) followed by BINAP (0.12 g, 0.19 mmol), palladium acetate(0.02 g, 0.09 mmol), and Cs₂CO₃ (2.7 g, 8.2 mmol). The reaction mixturewas stirred and N₂ was bubbled into it for 15 min. It was then heated at100° C. for 8 h under N₂ atmosphere. The reaction mixture was cooled toroom temperature, diluted with ethyl acetate (60 mL), washed with water(35 mL), brine (35 mL), and dried over Na₂SO₄. Concentration underreduced pressure offered a residue which was purified by columnchromatography (SiO₂, 60-120, product getting eluted inmethanol/chloroform: 8/92) to get 5 (0.50 g, 33%) as a white solid.

Step-3

To a solution of 5 (0.5 g, 1.1 mmol) in methanol (50 mL)) was added 10%Pd/C (0.05 g, 10% w/w) and the reaction mixture was allowed to stirunder H₂ atmosphere (1.5 Kg hydrogen pressure) at rt for 16 h. Thereaction mixture was filtered through a pad of celite and concentratedunder reduced pressure to get 6 (0.3 g, 65%) as a colorless viscousliquid.

Step-4

To a stirred solution of 6 (0.21 g, 0.5 mmol) and potassium carbonate(0.27 g, 2.0 mmol) in NMP (2.5 mL) at 0° C. was added acryloyl chloride(0.053 g, 0.6 mmol) and the reaction mixture was stirred at 0° C. for 30min The reaction mixture was added drop wise to a cold, stirringsolution of 10% NaHCO₃ and stirred at the same temperature (0° C.) for30 min. A white solid precipitated out which was isolated by filtrationthrough a Buchner funnel. The solid was washed with cold water andhexane and dissolved in mixture of methanol/dichloromethane (50:50, 10mL) and concentrated under reduced pressure. The residue obtained wassuspended in cold water (25 mL), Et₃N was added to it and it wasextracted with ethyl acetate (2×50 mL). The combined ethyl acetateextract was washed with water (50 mL), brine (50 mL), dried over Na₂SO₄and concentrated under reduced pressure to get I-184 (0.150 g, 65%) aswhite solid. ¹H NMR (DMSO-d₆) δ ppm: 1.89 (quin, J=7.2 Hz, 2H), 2.21 (t,J=7.6 Hz, 2H), 3.39 (t, J=7.2 Hz, 2H), 3.49 (t, J=5.2 Hz, 2H), 3.87 (t,J=5.6 Hz, 2H), 5.77 (dd, J=1.6 & 10.4 Hz, 1H), 6.26 (dd, J=1.6 & 17.2Hz, 1H), 6.39-6.46 (m, 2H), 6.95 (t, J=8.4 Hz, 1H), 7.03-7.12 (m, 3H),7.44 (t, J=8.4 Hz, 1H), 7.56 (d, J=8.4 Hz, 1H), 7.70 (s, 1H), 8.51 (d,J=2.8 Hz, 1H), 9.56 (s, 1H), 10.35 (s, 1H); LCMS: m/e 478 (M+1).

The intermediate 3-(2-(2-oxopyrrolidin-1-yl)ethoxyaniline 4 was preparedaccording to the scheme shown below.

A) NaH, THF, rt, 16 h; B) Pd—C, H₂, methanol, rt, 16 h.

Step-1

To a stirring solution of NaH (3.4 g, 141.6 mmol, 60% dispersion inparaffin oil) in dry THF (50 mL) was added 1 (6 g, 46.0 mmol) at 0° C.and the reaction mixture was stirred at rt for 15 min under nitrogenatmosphere. To it was added a solution of 2 (5.0 g, 35.4 mmol) in THF(10 mL) and the reaction mixture was stirred at rt for 16 h. It wasquenched with cold water (40 mL), and extracted with ethyl acetate (35mL). The ethyl acetate extract was washed with water (2×25 mL), brine(25 mL), dried over Na₂SO₄ and concentration under reduced pressure toget a residue which was purified by column chromatography (SiO₂, 60-120,product getting eluted in methanol/chloroform: 10/90) to get 3 (2.5 g,30%) as a brownish liquid.

Step-2

To a solution of 3 (2.2 g, 8.8 mmol) in methanol (50 mL)) was added 10%Pd/C (0.22 g, 10% w/w) and the reaction mixture was allowed to stirunder H₂ atmosphere (1.5 Kg hydrogen pressure) at rt for 16 h. Thereaction mixture was filtered through a pad of Celite® and concentratedunder reduced pressure to get 4 (1.7 g, 89%) as a yellowish liquid. Itwas used in the next step without further purification.

Example 104 Preparation ofN-(3-(2-(6-methoxypyridin-3-ylamino)-5-methylpyrimidin-4-yloxy)phenyl)acrylamideI-186

The title compound was prepared according to the schemes, steps andintermediates described in Example 103 by using2,4-dichloro-5-methylpyrimidine in place of 1 in Step-1 and6-methoxy-3-aminopyridine in place of 4 in Step-2. ¹H NMR (DMSO-d₆) δppm: 2.16 (s, 3H), 3.73 (s, 3H), 5.76 (dd, J=1.92 & 10.04 Hz, 1H), 6.24(dd, J=1.92 & 16.92 Hz, 1H), 6.39-6.49 (m, 2H), 6.92 (dd, J=1.48 & 8 Hz,1H), 7.40 (t, J=8.08 Hz, 1H), 7.49 (d, J=8.16 Hz, 1H), 7.64 (d, J=1.84Hz, 1H), 7.77-7.79 (m, 1H), 8.17 (bs, 1H), 8.20 (s, 1H), 9.27 (s, 1H),10.29 (s, 1H); LCMS: m/e 378 (M+1).

Example 105 Preparation ofN-(3-(5-methyl-2-(phenylamino)pyrimidin-4-yloxy)phenyl)acrylamide I-248

The title compound was prepared according to the schemes, steps andintermediates described below.

A) K₂CO₃, DMF, rt, 24 h; A′) (Boc)₂O, THF, 60° C., 2 h; B) aniline,conc. HCl, EtOH, 80° C., 1 h; C) TFA, CH₂Cl₂, 0° C. to rt, ½ h; D)acryloyl chloride, NMP, 0° C., 10 min.

Step-1

To a stirring solution of 2 (100 mg, 0.48 mmol) and K₂CO₃ (99.2 mg,0.717 mmol) in dry DMF (5 mL) was added 1 (78 mg, 0.478 mmol) and thereaction was continued at rt for 24 h under nitrogen atmosphere. Thereaction mixture was concentrated under reduced pressure and the residuewas diluted with ethyl acetate (10 mL). It was washed with water (2×5mL), brine (5 mL), dried over Na₂SO₄ and concentrated under reducedpressure to get 3 (120 mg, 75%) as a white solid. It was used for nextstep without further purification.

Step-2

A pressure tube was charged with 3 (75 mg, 0.224 mmol), conc. HCl (40mg, 0.4 mmol), aniline (83 mg, 0.89 mmol) and ethanol (2.0 mL). The tubewas screw capped and the contents were stirred at 80° C. for 60 min. Thereaction mixture was cooled, concentrated under reduced pressure and theresidue was quenched with water (5.0 mL). It was basified with 10%NaHCO₃ soln. and extracted with Ethyl acetate (3×10 mL). The combinedEtOAc layer was washed with water (2×5 mL), brine (5 mL), dried overNa₂SO₄ and concentrated under reduced pressure. The residue obtained wasfurther purified by column chromatography (SiO₂, 60-120 mesh,EtoAc/Hexane: 50/50) to get 4 (0.04 g, 45.9%) as an off-white sold.

Step-3

To a stirring solution of 4 (160 mg, 0.40 mmol) in dichloromethane (4.0mL) was added at 0° C., trifluoroacetic acid (0.8 mL). Stirring wascontinued at the same temperature for 30 min after which the reactionmixture was concentrated under reduced pressure and the residue wasdissolved in water (5.0 mL), basified with 10% NaHCO₃ solution andextracted with dichloromethane (2×5 mL). The dichloromethane extract waswashed with water (5 mL), brine (5 mL), dried over Na₂SO₄ andconcentrated under reduced pressure to get 5 (110 mg, 93.2%) as an offwhite solid. It was used for next step without further purification.

Step-4

To a stirred solution of 5 (75 mg, 0.256 mmol) in NMP (0.8 mL) at 0° C.was added acryloyl chloride (34.8 mg, 0.38 mmol) and the reactionmixture was stirred at 0° C. for 10 min. The reaction mixture wasquenched with water (4.0 mL), basified with 10% NaHCO3 soln. andextracted with dichloromethane (2×5 mL). The dichloromethane extract waswashed with water (5 mL), brine (5 mL), dried over Na₂SO₄ andconcentrated under reduced pressure. The residue obtained was furtherpurified column chromatography (SiO₂, 60-120 mesh, CHCl₃/MeOH: 99/1) toget I-248 (0.035 g, 39.6%) as a white colored solid. ¹H NMR (DMSO-d₆) δppm: 2.15 (s, 3H), 5.75 (dd, J=1.92 & 10.04 Hz, 1H), 6.24 (dd, J=1.96 &16.96 Hz, 1H), 6.41 (dd, J=10.6 & 17 Hz, 1H), 6.78-6.8 (m, 1H), 6.94(dd, J=1.44 & 8.04 Hz, 1H), 7.00 (t, J=7.52 Hz, 2H), 7.40-7.44 (m, 3H),7.53 (d, J=8.24 Hz, 1H), 7.6 (t, J=2 Hz, 1H), 8.23 (d, J=1.04 Hz, 1H),9.36 (s, 1H), 10.30 (s, 1H); LCMS: m/e 346.8 (M+1).

Example 106 Preparation of1-(4-(5-fluoro-2-(phenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-229

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using1-tert-butyloxycarbonyl-4-aminopiperidine in place of 2 in Step-1 andaniline in place of 4 in Step-2. ¹H NMR (DMSO-d₆) δ ppm: 1.35-1.50 (m,2H), 1.90-2.05 (m, 2H), 2.7-2.85 (m, 1H), 3.10-3.20 (m, 1H), 4.11-4.15(m, 2H), 4.46 (bd, J=13.72 Hz, 1H), 5.67 (dd, J=2.44 & 10.4 Hz, 1H),6.10 (dd, J=2.44 & 16.6 Hz, 1H), 6.82-6.88 (m, 2H), 7.22 (t, J=7.44 Hz,2H), 7.35 (d, J=7.56 Hz, 1H), 7.70 (d, J=7.72 Hz, 2H), 7.87 (d, i=3.76Hz, 1H), 9.07 (s, 1H); LCMS: m/e 341.383 (M+1).

Example 107 Preparation of2-((3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)(hydroxy)methyl)acrylonitrileI-71

The title compound was prepared according to the schemes, steps andintermediates described below.

A) 2, DIPEA, n-BuOH, 120° C., 12 h; B) conc. HCl, ethanol, 100° C., 5 h;C) LiOH, MeOH/THF/H2O, rt, 6 h; D) Me-NH—OMe.HCl, EDCI.HCl, HOBT, DIPEA,DMF, rt, 8 h; E) LAH (1.0 M soln. in THF), −78° C., 30 min; F) DABCO,1,4-dioxan/water, rt, 48 h.

Step-1

A solution of 1 (0.50 g, 2.99 mmol), 2 (0.45 g, 2.99 mmol) and DIPEA(0.57 g, 4.48 mmol) in n-butanol (5.0 mL) was heated in a pressure tube(120° C., 16 h). It was cooled, quenched with water (5 mL) and extractedwith EtOAc (2×5 mL). The combined EtOAc extract was washed with water (2mL), brine (2 mL), dried over Na₂SO₄ and concentrated under reducedpressure to afford 3 (0.70 g, 83.3%) as an off-white solid.

Step-2

A solution of 3 (0.5 g, 1.77 mmol) and 4 (0.29 g, 1.77 mmol) in ethanol(2.5 mL) was taken in a pressure tube and acetic acid (0.1 mL) was addedto it. The tube was tightly closed and the contents were stirred at 100°C. for 5 h. The reaction mixture was cooled, ethanol was removed underreduced pressure and the residue was taken in ethyl acetate (50 mL). Itwas washed with NaHCO₃ solution (5 mL), brine (5 mL), dried over Na₂SO₄and concentrated under reduced pressure. The solid precipitated wasisolated by filtration. It was dried under vacuum to get 5 (0.6 g, 80%).

Step-3

To a stirred solution of 5 (0.6 g, 1.4 mmol) in methanol/THF/water: 6mL/6 mL/3 mL was added LiOH (0.298 g, 7 mmol) and the reaction mixturewas stirred at rt for 2 h. It was concentrated under reduced pressure;residue was diluted with water (2 mL) and extracted with diethyl ether(5 mL). The aqueous layer was separated and acidified with 1.5 N HCl(pH˜4-5), concentrated and dried under vacuum to get 6 (0.4 g, 70%) as awhite solid which was taken for next step without further purification.

Step-4

To a stirred solution of 6 (0.4 g, 1 mmol) in DMF (3 mL) were addedMeNH-OMe.HCl (0.102 g, 0.1 mmol), EDCI.HCl (0.003 g, 1.5 mmol), HOBT (71mg, 0.5 mmol) and DIPEA (0.204 g, 1.5 mmol). The reaction mixture wasstirred at room temperature for 8 h and quenched with water andextracted with EtOAc (2×5 mL). The combined organic layer was washedwith brine, dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to get 7 (0.4 g, 90.9%) as a white solid.

Step-5

To a stirred solution of 7 (0.4 g, 0.9 mmol) in THF (10 mL) was addedLAH (1.8 mL, 1.8 mmol) at −78° C. The reaction mixture was allowed tostir at the same temperature for 30 mins after which it was quenchedwith Na₂SO₄ solution (2 mL) and extracted with ethyl acetate (10 mL).The ethyl acetate layer was separated and washed with water (2 mL),brine solution (2 mL) and dried over anhydrous Na₂SO₄. Filtrationfollowed by concentration under reduced pressure offered a residue whichwas purified by column chromatography (SiO₂, 60-120, pet ether/ethylacetate 7/3) to get 8 (200 mg, 58%) as a yellow solid.

Step-6

To a stirred solution of 8 (200 mg, 0.523 mmol) and 9 (69 mg, 1.3 mmol)in 1,4-dioxane/H₂O (1.4 mL/0.6 mL) was added DABCO (50 mg, 0.2523 mmol)at rt. Stirring was continued at room temperature for 48 h after whichthe reaction mixture was concentrated under reduced pressure. Theresidue obtained was further purified by column chromatography (SiO₂,pet ether/ethyl acetate, 6/4) to get I-71 as greenish gummy material(0.05 g, 22.7%). ¹H NMR (DMSO-d₆) δ ppm: 3.48 (s, 3H), 3.77 (t, J=4.4Hz, 2H), 4.11-4.16 (m, 2H), 5.11 (s, 1H), 5.99 (s, 1H), 6.06 (s, 1H),6.85 (s, 1H), 6.91 (d, J=8.84 Hz, 2H), 7.15 (d, J=7.44 Hz, 1H),7.30-7.40 (m, LCMS: m/e (M+1).

Example 108 Preparation of2-((4-(5-fluoro-2-(phenylamino)pyrimidin-4-ylamino)phenyl)(hydroxy)methyl)acrylonitrileI-161

The title compound was prepared according to the schemes, steps andintermediates described in Example 107 by using aniline in place of 4 inStep-2. ¹H NMR (CDCl₃) δ ppm: 5.32 (s, 1H), 6.07 (d, J=0.8 Hz, 1H), 6.15(d, J=1.6 Hz, 1H), 6.84 (d, J=2.8 Hz, 1H), 7.03-7.06 (m, 2H), 7.29 (t,J=1.6 Hz, 2H), 7.38 (d, J=8.44 Hz, 2H), 7.52 (d, J=8.8 Hz, 2H), 7.67(dd, J=1.6 & 6.4 Hz, 2H), 7.96 (d, J=3.2 Hz, 1H); LCMS: m/e 361.8 (M+1).

Example 109 Preparation of2-((4-(5-fluoro-2-(3-trifluoromethoxyphenylamino)pyrimidin-4-ylamino)phenyl)(hydroxy)methyl)acrylonitrileI-163

The title compound was prepared according to the schemes, steps andintermediates described in Example 107 by using3-trifluoromethoxyaniline in place of 4 in Step-2. ¹H NMR (DMSO-d₆) δppm: 5.29 (d, J=3.8 Hz, 1H), 6.13 (s, 1H), 6.19 (s, 1H), 6.31 (dd, J=3.8Hz, 1H), 6.83 (d, J=7.76 Hz, 1H), 7.11 (d, J=7.8 Hz, 1H), 7.30-7.37 (m,2H), 7.61-7.63 (m, 2H), 7.81 (s, 1H), 7.90 (d, J=7.4 Hz, 1H), 8.16 (dd,J=1.44 & 3.56 Hz, 1H), 9.45 (s, 1H), 9.53 (s, 1H); LCMS: m/e 446 (M+1).

Example 110 Preparation of N-(3-(5-fluoro-2-(3-(3-(methylsulfonyl)propoxy)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamide I-116

The title compound was prepared according to the schemes, steps andintermediates described in Example 98 by using3-(3-methylsulfonyl)propoxyaniline in place of 4 in Step-2. ¹H NMR(DMSO-d₆) δ ppm: 2.02-2.15 (m, 2H), 3.01 (s, 3H), 3.22 (t, J=7.56 Hz,2H), 3.88 (t, J=6.12 Hz, 2H), 5.77 (dd, J=1.84 & 10.12 Hz, 1H), 6.25(dd, J=1.72 & 16.88 Hz, 1H), 6.43 (d, J=9.96 & 16.76 Hz, 2H), 6.95 (t,J=8.12 Hz, 1H), 7.06 (t, J=7.48 Hz, 2H), 7.13 (s, 1H), 7.44 (t, J=8.12Hz, 1H), 7.56 (d, J=8.44 Hz, 1H), 7.68 (s, 1H), 8.50 (d, J=2.84 Hz, 1H),9.57 (s, 1H), 10.34 (s, 1H); LCMS: m/e 487.0 (M+2).

Example 111 Preparation ofN-(3-(5-cyclopropyl-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamideI-131

The title compound was prepared according to the steps and intermediatesas described below.

A) K₂CO₃, DMA, rt, 5 h; B) PTSA, dioxane, 100 ⁰C, 2 h; C) potassiumcyclopropyltrifluoroborate, Pd(OAc)₂, Xanphos, Cs₂CO₃, toluene, 100° C.,12 h; D) 4N HCL, dioxane, rt, 1 hr; then acryoyl chloride, Et₃N, DCM,−10° C., 10 min

Step-1

To a solution of 5-bromo-2,4-dichloropyrimidine (0.68 g, 3.0 mmol) andtert-butyl 3-hydroxyphenylcarbamate (0.65 g, 3.1 mmol) in DMA (4 mL) wasadded K₂CO₃ (0.83 g, 6.0 mmol). The suspension was stirred for 5 hours.Water (15 ml) was added and the precipitate was collected by filtration.The solid was washed with ether and dried to yield 1.2 g of compound 3.MS: m/e=400.2, 402.2 (M+1).

Step-2

To a solution of compound 3 (200 mg, 0.5 mmol) and4-(2-methoxyethoxy)aniline (0.1 g, 0.6 mmol) in 5 ml dioxane was added4-methylbenzenesulfonic acid monohydrate (0.08 g, 0.4 mmol). The mixturewas stirred at 100° C. for two hours. The solvent was evaporated. Theresidue was dissolved in 20 ml ethyl acetate and washed with NaHCO₃aqueous solution, water and brine. The organic layer was separated anddried over Na₂SO₄. After removal of solvent, the crude product wassubject to chromatography on silica gel (hexane:EtOAc=1:1). 0.10 g ofcompound 5 was obtained: MS m/z: 531.1, 531.0 (M+H⁺).

Step-3

Potassium cyclopropyltrifluoroborate (36 mg, 0.25 mmol), compound 5(0.10 g, 0.19 mmol), palladium acetate (3.4 mg, 0.015 mmol), Xantphos(17.5 mg, 0.03 mmol) and Cs₂CO₃ (186 mg, 0.57 mmol) were suspended in 5mL toluene and 1 mL water. The mixture was degassed, sealed under argonand heated at 100° C. for 12 hours. 20 mL ethyl acetate was added andwashed with NaHCO₃ aqueous solution, water and brine. The organic layerwas separated and dried over Na₂SO₄. After removal of solvent, the crudeproduct was subject to chromatography on silica gel (hexane:EtOAc=1:1).50 mg of compound 6 was obtained: MS m/z: 493.2 (M+H⁺).

Step-4

The title compound was prepared from compound 6 following the proceduredescribed in Example 96. MS m/z: 447.1 (M+H⁺).

Example 112 Preparation of1-(4-(5-fluoro-2-(3-(2-dimethylaminoethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-2-methylprop-2-en-1-oneI-207

The title compound was prepared according to the schemes steps andintermediates described below.

A) 2, DIPEA, n-BuOH, 90° C., 12 h; B) 4, Pd(OAc)₂, BINAP, Cs₂CO₃,toluene, 110° C., 16 h; C) LiOH, MeOH/THF/H2O, rt, 6 h; D) MeNHOMe.HCl,EDCI.HCl, HOBT, DIPEA, DMF, rt, 3 h; E) 8, THF, 0° C. to rt, 2 h.

Step-1

A solution of 1 (4 g, 23.9 mmol), 2 (3.6 g, 23.7 mmol) and DIPEA (4.6 g,35.58 mmol) in n-butanol (40 mL) was heated in a pressure tube (90° C.,12 h). It was cooled, quenched with water (5 mL) and extracted withEtOAc (2×5 mL). The combined EtOAc extract was washed with water (60mL), brine (40 mL), dried over Na₂SO₄ and concentrated under reducedpressure to afford 3 (5.5 g, 82%) as an off-white solid.

Step-2

A solution of 4 (0.319 g, 1.76 mmol), 3 (0.5 g, 1.76 mmol), Pd(OAc)₂(0.039 g, 0.17 mmol), BINAP (0.055 g, 0.08 mmol) and Cs₂CO₃ (1.44 g,4.42 mmol) in degassed toluene (toluene was purged with N₂ for 30 min)was heated for 16 h at 110° C. under N₂ atmosphere. The reaction mixturewas cooled, diluted with EtOAc (25 mL) and washed with water (5 mL),brine (2 mL) and dried over Na₂SO₄. Filtration followed by concentrationunder reduced pressure offered a residue which was further purified bycolumn chromatography (SiO₂, 60-120, chloroform/methanol, 9/1) to get 4(0.63 g, 84%) as yellow solid.

Step-3

To a stirred solution of 5 (0.3 g, 0.70 mmol) in methanol/THF/water: 1mL/1 mL/0.5 mL was added LiOH (0.147 g, 3.52 mmol) and the reactionmixture was stirred at rt for 6 h. It was concentrated under reducedpressure; residue was diluted with water (2 mL) and extracted withdiethyl ether (5 mL). The aqueous layer was separated and acidified with1.5 N HCl (pH˜4-5) which was concentrated as such and dried under vacuumto get 6 (0.31 g, crude) as yellow gummy solid which was taken for nextstep without further purification.

Step-4

To a stirred solution of 6 (0.29 g, 0.70 mmol) in DMF (3 mL) were addedMeNH-OMe.HCl (0.068 g, 0.70 mmol), EDCI.HCl (0.202 g, 1.05 mmol), HOBT(0.047 g, 0.35 mmol) and DIPEA (0.136 g, 1.05 mmol). The reactionmixture was stirred at room temperature for 3 h, quenched with water andextracted with EtOAc (2×5 mL). The combined organic layer was washedwith brine, dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. The residue was further purified by columnchromatography (SiO₂, 60-120, methanol/chloroform: 20/80) to get 7(0.061 g, 19%) as gummy yellow solid.

Step-5

To a stirred solution of 7 (100 mg, 0.22 mmol) in THF (1 mL) at 0° C.was added 8 (17.6 mL, 8.80 mmol). The reaction mixture was allowed tostir at room temperature for 2 h. It was quenched with saturated NH₄Clsolution (0.5 mL) and extracted with EtOAc (2×3 mL). The combinedorganic layer was washed with brine, dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to get a white solid.It was further purified by column chromatography (SiO₂, 60-120, productgetting eluted in 20% methanol/chloroform) to get I-207 (9 mg. 9%) asgummy yellow solid. ¹H NMR (DMSO-d₆) δ ppm: 1.90 (s, 3H), 2.01 (s, 3H),2.19 (s, 6H), 2.57 (t, J=5.64 Hz, 2H), 3.87 (t, J=5.84 Hz, 2H), 6.45(dd, J=1.64 & 8.08 Hz, 1H), 6.82 (s, 1H), 7.04 (t, J=8.16 Hz, 1H), 7.18(d, J=8.16 Hz, 1H), 7.33 (s, 1H), 7.47 (t, J=7.88 Hz, 1H), 7.62 (d,J=7.72 Hz, 1H), 8.13-8.16 (m, 3H), 9.24 (s, 1H), 9.56 (s, 1H); LCMS: m/e450.1 (M+1).

Example 113 Preparation of1-(4-(5-fluoro-2-(phenylamino)pyrimidin-4-ylamino)phenyl)-3-methylbut-2-en-1-oneI-206

The title compound was prepared according to the schemes, steps andintermediates described in Example 112 by using methyl 4-aminobenzoatein place of 2 in step-1 and aniline in place of 4 in step-2. ¹H NMR(DMSO-d₆) δ ppm: 1.98 (s, 3H), 2.12 (s, 3H), 6.90-7.00 (m, 2H),7.20-7.30 (m, 2H), 7.65 (d, J=8.16 Hz, 2H), 7.90 (d, J=8.56 Hz, 2H),7.98 (d, J=8.68 Hz, 2H), 8.18 (bs, 1H), 9.31 (s, 1H), 9.68 (s, 1H);LCMS: m/e 363.0 (M+1).

Example 114 Preparation of1-(3-(5-fluoro-2-(3-(prop-2-ynyloxy)phenylamino)pyrimidin-4-ylamino)phenyl)-3-methylbut-2-en-1-oneI-211

The title compound was prepared according to the schemes, steps andintermediates described in Example 112 by using 3-prop-2-ynyloxyanilinein place of 4 in step-2. ¹H NMR (CD₃OD) δ ppm: 2.0 (d, J=1 Hz, 3H), 2.21(d, J=1.04 Hz, 3H), 2.94-2.96 (d, J=2.44 Hz, 1H), 4.59 (d, J=2.36 Hz,2H), 6.79-6.81 (m, 2H), 7.03 (dd, J=3.12 & 8.04 Hz, 1H), 7.14 (t, J=2.2Hz, 1H), 7.23 (t, J=8.12 Hz, 1H), 7.54 (t, J=7.92 Hz, 1H), 7.83 (d,J=7.96 Hz, 1H), 7.86 (dd, J=2.08 & 8.08 Hz, 1H), 8.03 (d, J=4.96 Hz,1H), 8.21 (t, J=1.88 Hz, 1H); LCMS: m/e 417.0 (M+1).

Example 115 Preparation of1-(3-(5-fluoro-2-(3-(trifluoromethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-3-methylbut-2-en-1-oneI-223

The title compound was prepared according to the schemes, steps andintermediates described in Example 112 by using3-trifluoromethoxyaniline in place of 4 in step-2. ¹H NMR (CDCl₃) δ ppm:2.0 (d, J=1.08 Hz, 3H), 2.24 (d, J=1.04 Hz, 3H), 6.74 (t, J=1.24 Hz,1H), 6.85 (dd, J=1.08 & 7.0 Hz, 1H), 6.90 (s, 1H), 7.08 (s, 1H),7.24-7.28 (m, 1H), 7.35 (td, J=1.2 & 7.44 Hz, 1H), 7.49 (t, J=7.88 Hz,1H), 7.63 (s, 1H), 7.72 (td, J=1.04 & 7.76 Hz, 1H), 7.90-7.92 (m, 1H),8.00-8.05 (m, 2H); LCMS: m/e 447 (M+1).

Example 116 Preparation of1-(3-(5-fluoro-2-(3-(trifluoromethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-2-methylprop-2-en-1-oneI-199

The title compound was prepared according to the schemes, steps andintermediates described in Example 112 by using3-trifluoromethoxyaniline in place of 4 in step-2 andisopropenylmagnesium bromide in place of 8 in step-5. ¹H NMR (DMSO-d₆) δppm: 1.97 (s, 3H), 5.6 (s, 1H), 6.0 (d, J=0.96 Hz, 1H), 6.82 (d, J=8.08Hz, 1H), 7.27 (t, J=8.2 Hz, 1H), 7.41 (dd, J=1.12 & 7.56 Hz, 1H), 7.48(t, J=7.76 Hz, 1H), 7.60 (dd, J=1.28 & 7.88 Hz, 1H), 7.78 (s, 1H), 7.90(d, J=1.64 Hz, 1H), 8.15 (d, J=8 Hz, 1H), 8.19 (d, J=3.64 Hz, 1H), 9.55(s, 1H), 9.65 (s, 1H); LCMS: m/e 433 (M+1).

Example 117 Preparation of1-(4-(5-fluoro-2-(phenylamino)pyrimidin-4-ylamino)phenyl)-2-methylprop-2-en-1-oneI-185

The title compound was prepared according to the schemes, steps andintermediates described in Example 112 by using methyl 4-aminobenzoatein plave of 2 in step-1, aniline in place of 4 in step-2 andisopropenylmagnesium bromide in place of 8 in step-5. ¹H NMR (DMSO-d₆) δppm: 1.99 (s, 3H), 5.54 (s, 1H), 1.01 (s, 1H), 6.93 (t, J=7.36 Hz, 1H),7.24 (t, J=7.52 Hz, 2H), 7.66-7.72 (m, 4H), 8.01 (d, J=8.72 Hz, 2H),8.19 (d, J=3.6 Hz, 1H), 9.32 (s, 1H), 9.72 (s, 1H); LCMS: m/e 348.8(M+1).

Example 118 Preparation ofN-(3-(2-(3-(2-(dimethylamino)ethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-233

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using3-(2-dimethylaminoethoxy)aniline in place of 4 in step-2. ¹H NMR (CD₃OD)δ ppm: 2.31 (s, 6H), 2.76 (t, J=5.6 Hz, 2H), 3.97 (t, J=5.2 Hz, 2H),5.78 (dd, J=2 & 9.2 Hz, 1H), 6.38-6.42 (m, 2H), 6.52-6.55 (m, 1H),7.1-7.11 (m 2H), 7.30 (t, J=8.0 Hz, 1H), 7.36 (s, 1H), 7.42-7.48 (m,2H), 7.94 (d, J=3.6 Hz, 1H), 8.05 (s, 1H); LCMS: m/e 437 (M+1).

Example 119 Preparation ofN-(3-(5-fluoro-4-(4-phenoxyphenoxy)pyrimidin-2-ylamino)phenyl)acrylamideI-130

The title compound was prepared according to the steps, schemes andintermediates described in Example 11 by using5-fluoro-2,4-dichloropyrimidine in place of 1 in step-1. ¹H NMR(DMSO-d₆) δ ppm: 5.71 (dd, J=1.6 & 10 Hz, 1H), 6.22 (dd, J=1.6 & 16.8Hz, 1H), 6.44 (dd, J=10.4 & 17.2 Hz, 1H), 6.98-7.05 (m, 3H), 7.1-7.12(m, 2H), 7.17 (t, J=7.2 Hz, 1H), 7.24 (t, J=7.6 Hz, 2H), 7.35-7.37 (m,2H), 7.42 (t, J=8.4 Hz, 2H), 7.71 (s, 1H), 8.5 (s, 1H), 9.6 (s, 1H),10.05 (s, 1H); LCMS: m/e 443.0 (M+1).

Example 120 Preparation of(S)—N-(3-(5-fluoro-2-(4-(tetrahydrofuran-3-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-43

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using(S)-4-(tetrahydrofuran-3-yloxyaniline in place of 4 in step-2. ¹H NMR(DMSO-d₆, 500 MHz): δ 10.10 (s, 1H), 9.35 (s, 1H), 8.95 (s, 1H), 8.05(d, J=4.0 Hz, 1H), 7.92 (s, 1H), 7.52 (d, J=9.0 Hz, 2H), 7.47 (d, J=7.5Hz, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.27 (t, J=8.0 Hz, 1H), 6.72 (d, J=9.0Hz, 2H), 6.45 (dd, J=1.5, 17.0 Hz, 1H), 6.25 (dd, J=1.1, 16.5 Hz, 1H),5.75 (dd, J=1.1, 10.0 Hz, 1H), 4.93-4.84 (m, 1H), 3.88-3.72 (m, 4H),2.20-2.10 (m, 1H), 1.97-1.90 (m, 1H). MS m/e=436 [M⁺+1]

Example 121 Preparation of(R)—N-(3-(5-fluoro-2-(4-(tetrahydrofuran-3-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-46

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using(R)-4-(tetrahydrofuran-3-yloxyaniline in place of 4 in step-2. ¹H NMR(DMSO-d₆, 500 MHz): δ 10.10 (s, 1H), 9.35 (s, 1H), 8.95 (s, 1H), 8.05(d, J=4.0 Hz, 1H), 7.92 (s, 1H), 7.52 (d, J=9.0 Hz, 2H), 7.47 (d, J=7.5Hz, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.27 (t, J=8.0 Hz, 1H), 6.72 (d, J=9.0Hz, 2H), 6.45 (dd, J=1.5, 17.0 Hz, 1H), 6.25 (dd, J=1.1, 16.5 Hz, 1H),5.75 (dd, J=1.1, 10.0 Hz, 1H), 4.93-4.84 (m, 1H), 3.88-3.72 (m, 4H),2.22-2.14 (m, 1H), 1.97-1.90 (m, 1H). MS: m/e=436 [M⁺+1]

Example 122 Preparation ofN-(3-(5-fluoro-2-(3-((1-methylpiperidin-3-yl)methoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-acrylamideI-76)

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using3-(1-methylpiperidin-3-yl)methoxyaniline in place of 4 in step-2. ¹H NMR(DMSO-d₆, 500 MHz): δ 10.08 (s, 1H), 9.41 (s, 1H), 9.09 (s, 1H), 8.11(d, J=3.5 Hz, 1H), 7.90 (s, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.41 (d, J=8.0Hz, 1H), 7.32 (s, 1H), 7.30-7.20 (m, 2H), 7.03 (t, J=8.0 Hz, 1H),6.50-6.40 (m, 2H), 6.24 (dd, J=1.5, 17.0 Hz, 1H), 5.74 (dd, J=2.0, 10.5Hz, 1H), 3.75-3.65 (m, 2H), 2.73 (d, J=10 Hz, 1H), 2.60 (d, J=10.5 Hz,1H), 2.13 (s, 3H), 1.98-1.83 (m, 2H), 1.75-1.58 (m, 3H), 1.55-1.45 (m,1H), 1.05-0.95 (m, 1H). MS: m/e=477 (M⁺+1).

Example 123 Preparation ofN-(3-(5-fluoro-2-(3-((1-methylpiperidin-4-yl)methoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-acrylamideI-82

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using3-(1-methylpiperidin-4-yl)methoxyaniline in place of 4 in step-2. ¹H-NMR(CDCl₃+DMSO-D₆, 500 MHz): δ 9.04 (bs, 1H), 8.30 (s, 1H), 7.96 (s, 1H),7.75 (s, 1H), 7.63 (s, 1H), 7.59 (d, J=7.0 Hz, 1H), 7.38-7.33 (m, 2H),7.27 (t, J=8.5 Hz, 1H), 7.16 (t, J=8 Hz, 1H), 7.09 (d, J=8.5 Hz, 1H),6.52 (d, J=7.0 Hz, 1H), 6.51-6.38 (m, 2H), 5.73 (dd, J=2.0, 9.0 Hz, 1H),3.77 (d, J=6.0 Hz, 2H), 2.90-2.84 (m, 2H), 2.28 (s, 3H), 1.95 (t, J 10.0Hz, 1H), 1.85-1.74 (m, 2H), 1.45-1.32 (m, 2H), 1.28-1.25 (m, 2H). MS:m/e=477 (M⁺+1).

Example 124 Preparation ofN-(3-(2-(3-(4-(2-hydroxyethyl)piperazin-1-yl)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-83

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using3-(4-(2-t-butyldimethylsilyloxyethyl)piperazin-1-ylaniline in place of 4in step-2 and deprotecting the TBS ether with TFA in DCM as a step-5. ¹HNMR (DMSO-d₆, 500 MHz): δ 8.99 (s, 1H), 8.85 (s, 1H), 8.04 (d, J=4.0 Hz,1H), 7.28-7.19 (m, 2H), 7.08-7.00 (m, 2H), 6.94 (t, J=3.5 Hz, 2H), 6.48(dd, J=2.0, 8.0 Hz, 1H), 6.35-6.31 (m, 1H), 4.94 (s, 2H), 3.71 (t, J=6.0Hz, 2H), 3.02 (t, J=4.5 Hz, 4H), 2.57-2.50 (m, 4H), 2.46 (t, J=6.0 Hz,2H), 0.87 (s, 9H), 0.05 (s, 6H). MS: m/e=538 (M⁺+1).

Example 125 Preparation ofN-(4-(5-fluoro-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)benzyl)-N-methylacrylamideI-113

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using4-(N-methyl-N-tert-butyloxycarbonylamino)methylaniline in place of 2 instep-1 and 3-methoxyaniline in place of 4 in step-2. ¹H-NMR (DMSO-d₆,200 MHz): δ 9.36 (bs, 1H), 9.16 (bs, 1H), 8.10 (d, J=3.4 Hz, 1H),7.83-7.70 (m, 2H), 7.34 (bs, 1H), 7.26-7.01 (m, 4H), 6.86-6.73 (m, 1H),6.48 (d, J=8.0 Hz, 1H), 6.21 (dd, J=16.4, 2.2 Hz, 1H), 5.76-5.64 (m,1H), 4.64-4.53 (two s, 2H), 3.65 (s, 3H), 3.00-2.88 (two s, 3H). MS:m/e=408.2 [M⁺+1].

Example 126 Preparation ofN-(4-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)benzyl)-N-methylacrylamideI-114

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using4-(N-methyl-N-tert-butyloxycarbonylamino)methylaniline in place of 2 instep-1 and 6-methoxy-3-aminopyridine in place of 4 in step-2. ¹H-NMR(DMSO-D₆, 200 MHz): δ 9.36 (bs, 1H), 9.09 (bs, 1H), 8.32 (bs, 1H), 8.06(dd, J=3.8 Hz, 1H), 7.97-7.92 (m, 1H), 7.76-7.68 (m, 2H), 7.20-7.08 (m,2H), 6.87-6.75 (m, 1H), 6.72 (d, J=8.4 Hz, 1H), 6.22 (dd, J=19.4, 2.6Hz, 1H), 5.74-5.65 (m, 1H), 4.64-4.53 (two s, 2H), 3.78 (s, 3H),3.00-2.88 (two s, 3H). MS: m/e=409 (M⁺+1).

Example 127 Preparation ofN-(3-(5-fluoro-2-(3-methyoxyphenylamino)pyrimidin-4-ylamino)benzyl)-N-methylacrylamideI-115

The title compound was prepared according to the steps, schemes andintermediates described in Example 20 by using3-(N-methyl-N-tert-butyloxycarbonylamino)methylaniline in place of 2 instep-1 and 3-methoxyaniline in place of 4 in step-2. ¹H-NMR (DMSO-d₆,200 MHz): δ 9.50-9.30 (m, 1H), 9.15-8.96 (m, 1H), 8.15 (bs, 1H),7.82-7.59 (m, 2H), 7.45-7.00 (m, 4H), 6.97-6.65 (m, 2H), 6.55-6.45 (m,1H), 6.26-6.12 (m, 1H), 5.78-5.60 (m, 1H), 4.68 (s, 1H), 4.55 & 3.75(two s, 3H), 2.90 & 3.00 (two s, 3H). MS: m/e=408.2 [M⁺+1].

Example 128 Preparation ofN-(3-(5-fluoro-2-(3-(4-(2-hydroxyethyl)piperazin-1-yl)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamide I-84

The title compound was prepared according to the steps, schemes andintermediates described in Example 98 by using3-(4-(2-t-butyldimethylsilyloxyethyl)piperazin-1-ylaniline in place of 4in step-2 and deprotecting the TBS ether with TFA in DCM as a step-5.¹H-NMR (DMSO-D₆, 500 MHz): δ 8.19 (s, 1H), 7.75-7.70 (m, 2H), 7.42-7.35(m, 2H), 7.12-7.05 (m, 2H), 6.97 (dd, J=2.0, 10.5 Hz, 1H), 6.93 (s, 1H),6.72 (d, J=6.5 Hz, 1H), 6.57-6.54 (m, 1H), 6.44 (d, J=17.0 Hz, 1H),6.29-6.20 (m, 1H), 5.78 (d, J=10.0 Hz, 1H), 3.68 (t, J=5.5 Hz, 2H),3.00-2.94 (m, 4H), 2.63-2.56 (m, 6H). MS: m/e=479 (M++1).

Example 129 Preparation ofN-(3-(5-fluoro-2-(3-((1-methylpiperidin-3-yl)methoxy)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamideI-81

The title compound was prepared according to the steps, schemes andintermediates described in Example 98 by using3-(1-methylpiperidin-3-yl)methoxyaniline in place of 4 in step-2. ¹H-NMR(CDCl₃, 500 MHz): δ 8.19 (d, J=2.5 Hz, 1H), 7.82-7.75 (m, 2H), 7.42-7.36(m, 2H), 7.08-7.02 (m, 3H), 6.99 (d, J=7.0 Hz, 1H), 6.91 (s, 1H), 6.79(d, J=7.5 Hz, 1H), 6.48-6.44 (m, 1H), 6.42 (s, 1H), 6.29-6.23 (m, 1H),5.77 (d, J=10 Hz, 1H), 3.67-3.62 (m, 2H), 2.95-2.91 (m, 1H), 2.82-2.76(m, 1H), 2.28 (s, 3H), 2.11-2.05 (m, 1H), 1.98-1.92 (m, 1H), 1.79-1.70(m, 3H), 1.11-1.04 (m, 1H). MS: m/e=478 (M⁺+1).

Example 130 Preparation ofN-(3-(5-fluoro-2-(3-((1-methylpiperidin-4-yl)methoxy)phenylamino)pyrimidin-4-yloxy)phenyl)-acrylamideI-75

The title compound was prepared according to the steps, schemes andintermediates described in Example 98 by using3-(1-methylpiperidin-4-yl)methoxyaniline in place of 4 in step-2. ¹H-NMR(DMSO-D₆, 500 MHz): δ 10.31 (s, 1H), 9.50 (s, 1H), 8.50 (s, 1H), 7.67(s, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.42 (t, J=8.0 Hz, 1H), 7.10 (s, 1H),7.04 (d, J=7.0 Hz, 2H), 6.94 (t, J 8.0 Hz, 1H), 6.45-6.39 (m, 2H), 6.27(d, J=15.0 Hz, 1H), 5.78 (dd, J=2.0, 10.5 Hz, 1H), 3.64 (d, J=6.0 Hz,2H), 2.75 (d, J=6.5 Hz, 2H), 2.14 (s, 3H), 1.83 (t, J=10.5 Hz, 2H),1.66-1.64 (m, 3H), 1.25-1.23 (m, 2H). MS: m/e=478 (M⁺+1).

Example 131 Preparation ofN-(3-(5-cyano-2-(phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide I-157

The title compound was prepared according to the schemes, steps andintermediates described in Example 94, by using2,4-dichloro-5-cyanopyrimidine in the place of 4 in Step 2. MS 379.1(M+Na).

Example 132 Preparation ofN-(3-(5-fluoro-2-(3-(trifluoromethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-244

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-(trifluoromethoxy)aniline in the place of 4 in Step 2. MS 434.1 (M+1).

Example 133 Preparation ofN-(3-(5-fluoro-2-(pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-234

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using 3-aminopyridine in theplace of 4 in Step 2. MS 351.1 (M+1).

Example 134 Preparation ofN-(3-(5-fluoro-2-(4-fluorophenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-247

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using 4-fluoroaniline in theplace of 4 in Step 2. MS 368.1 (M+1)

Example 135 Preparation ofN-(3-(5-fluoro-2-(3-(3-morpholinopropoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-208

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-(3-morpholinopropoxy)aniline in the place of 4 in Step 2. MS 515.3(M+Na).

Example 136 Preparation ofN-(3-(2-(3-(3-(1H-imidazol-1-yl)propoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-204

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-(3-(1H-imidazol-1-yl)propoxy)aniline in the place of 4 in Step 2. MS474.3 (M+Na).

Example 137 Preparation ofN-(3-(2-(1-acetylpiperidin-3-ylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-238

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using1-(3-aminopiperidin-1-yl)ethanone in the place of 4 in Step 2. MS 421.1(M+Na).

Example 138 Preparation ofN-(3-(5-fluoro-2-(phenylamino)pyrimidin-4-yloxy)phenyl)acrylamide I-228

The title compound was prepared according to the schemes, steps andintermediates described in Example 98, by using aniline in the place of4 in Step 2. MS 351.3 (M+1).

Example 139 Preparation ofN-(3-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-243

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using 6-methoxypyridin-3-aminein the place of 4 in Step 2. MS 381.1 (M+1).

Example 140 Preparation ofN-(3-(5-methoxy-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)phenyl)acrylamide.I-158

The title compound was prepared according to the schemes, steps andintermediates described in Example 1, by using5-methoxy-2,4-dichloropyrimidine in the place of 1 in Step 1 and3-methoxyaniline in place of 4 in step 2. MS 392.3 (M+1).

Example 141 Preparation ofN-(3-(5-methoxy-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-192

The title compound was prepared according to the schemes, steps andintermediates described in Example 1, by using5-methoxy-2,4-dichloropyrimidine in the place of 1 in Step 1 and5-amino-2-methoxypyridine in place of 4 in step 2. MS 393.3 (M+1).

Example 142 Preparation ofN-(3-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-yloxy)phenyl)acrylamideI-222

The title compound was prepared according to the schemes, steps andintermediates described in Example 98, by using3-amino-6-methoxypyridine in the place of 4 in Step 2. MS 382.3 (M+1).

Example 143 Preparation of4-(3-acrylamidophenylamino)-N-tert-butyl-2-(6-methoxypyridin-3-ylamino)pyrimidine-5-carboxamide I-216

The title compound was prepared according to the schemes, steps andintermediates described in Example 37, by using tert-butylamine in theplace of 2 in Step-1 and omitting Step-6. MS 484.3 (M+Na).

Example 144 Preparation of(R)-1-(3-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-202

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using (R)-tert-butyl3-aminopiperidine-1-carboxylate in the place of 2 in Step 1 and3-amino-6-methoxypyridine in place of 4 in step 2. MS 395.3 (M+Na).

Example 145 Preparation of(R)-1-(3-(5-fluoro-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-195

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using (R)-tert-butyl3-aminopiperidine-1-carboxylate in the place of 2 in Step 1 and3-methoxyaniline in place of 4 in step 2. MS 394.3 (M+Na).

Example 146 Preparation of(S)-1-(3-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-197

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using (S)-tert-butyl3-aminopiperidine-1-carboxylate in the place of 2 in Step 1 and3-amino-6-methoxypyridine in place of 4 in step 2. MS 373.3 (M+1).

Example 147 Preparation of(S)-1-(3-(5-fluoro-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-196

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using (S)-tert-butyl3-aminopiperidine-1-carboxylate in the place of 2 in Step 1 and3-methoxyaniline in place of 4 in step 2. MS 372.3 (M+1).

Example 148 Preparation of(R)-1-(3-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-yloxy)piperidin-1-yl)prop-2-en-1-oneI-180

The title compound was prepared according to the schemes, steps andintermediates described in Example 98, by using (R)-tert-butyl3-hydroxypiperidine-1-carboxylate in the place of 2 in Step 1 and3-amino-6-methoxypyridine in place of 4 in step 2. MS 374.3 (M+1).

Example 149 Preparation of(R)-1-(3-(5-fluoro-2-(3-methoxyphenylamino)pyrimidin-4-yloxy)piperidin-1-yl)prop-2-en-1-oneI-190

The title compound was prepared according to the schemes, steps andintermediates described in Example 98, by using (R)-tert-butyl3-hydroxypiperidine-1-carboxylate in the place of 2 in Step 1 and3-methoxyaniline in place of 4 in step 2. MS 395.3 (M+Na).

Example 150 Preparation of(S)-1-(3-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-yloxy)piperidin-1-yl)prop-2-en-1-oneI-193

The title compound was prepared according to the schemes, steps andintermediates described in Example 98, by using (S)-tert-butyl3-hydroxypiperidine-1-carboxylate in the place of 2 in Step 1 and3-amino-6-methoxypyridine in place of 4 in step 2. MS 396.3 (M+Na).

Example 151 Preparation of(S)-1-(3-(5-fluoro-2-(3-methoxyphenylamino)pyrimidin-4-yloxy)piperidin-1-yl)prop-2-en-1-oneI-179

The title compound was prepared according to the schemes, steps andintermediates described in Example 98, by using (S)-tert-butyl3-hydroxypiperidine-1-carboxylate in the place of 2 in Step 1 and3-methoxyaniline in place of 4 in step 2. MS 395.3 (M+Na).

Example 152 Preparation of1-(3-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)pyrrolidin-1-yl)prop-2-en-1-oneI-203

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using tert-butyl3-aminopyrrolidine-1-carboxylatein the place of 2 in Step 1 and3-amino-6-methoxypyridine in place of 4 in step 2. MS 381.3 (M+Na).

Example 153 Preparation of1-(3-(5-fluoro-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)pyrrolidin-1-yl)prop-2-en-1-oneI-201

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using tert-butyl3-aminopyrrolidine-1-carboxylate in the place of 2 in Step 1 and3-methoxyaniline in place of 4 in step 2. MS 358.3 (M+1).

Example 154 Preparation of(R)-1-(3-(5-fluoro-2-(3-methoxyphenylamino)pyrimidin-4-ylthio)piperidin-1-yl)prop-2-en-1-oneI-137

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using (S)-tert-butyl3-mercaptopiperidine-1-carboxylate in the place of 2 in Step 1 and3-methoxyaniline in place of 4 in step 2. MS 411.1 (M+Na).

Example 155 Preparation of(R)-1-(3-(2-(3-chlorophenylamino)-5-fluoropyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-147

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using (S)-tert-butyl3-aminopiperidine-1-carboxylate in the place of 2 in Step 1 and3-chloroaniline in place of 4 in step 2. MS 376.1 (M+1).

Example 156 Preparation of(R)-1-(3-(5-fluoro-2-(3-(2-morpholinoethoxy)phenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-135

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using (S)-tert-butyl3-aminopiperidine-1-carboxylate in the place of 2 in Step 1 and3-(2-morpholinoethoxy)aniline in place of 4 in step 2. MS 471.3 (M+1).

Example 157 Preparation of(E)-4-(dimethylamino)-N-(3-(5-fluoro-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)but-2-enamideI-125

The title compound was prepared according to the schemes, steps andintermediates described in Example 139, by using(E)-4-(dimethylamino)but-2-enoyl chloride in the place of 7 in Step 4.MS 460.1 (M+Na).

Example 158 Preparation of2-((1H-pyrazol-1-yl)methyl)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)acrylamide.I-98

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using2-((1H-pyrazol-1-yl)methyl)acryloyl chloride in the place of 6 in Step3. MS 466.1 (M+Na).

Example 159 Preparation of(E)-4-(azetidin-1-yl)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)but-2-enamideI-123

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(E)-4-(azetidin-1-yl)but-2-enoyl chloride in the place of 6 in Step 3.MS 455.1 (M+Na).

Example 160 Preparation of(E)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)-4-morpholinobut-2-enamideI-102

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(E)-4-(morpholin-4-yl)but-2-enoyl chloride in the place of 6 in Step 3.MS 485.3 (M+Na).

Example 161 Preparation of(E)-4-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)but-2-enamideI-101

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(E)-4-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-2-yl)but-2-enoyl chloridein the place of 6 in Step 3. MS 496.1 (M+Na).

Example 162 Preparation of(E)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)-4-((2-methoxyethyl)(methyl)amino)but-2-enamideI-120

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(E)-4-((2-methoxyethyl)(methyl)amino)but-2-enoyl chloride in the placeof 6 in Step 3. MS 487.3 (M+Na).

Example 163 Preparation of(S,E)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)-4-(3-hydroxypyrrolidin-1-yl)but-2-enamideI-99

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(S,E)-4-(3-hydroxypyrrolidin-1-yl)but-2-enoyl chloride in the place of 6in Step 3. MS 485.3 (M+Na)

Example 164 Preparation of(R,E)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)-4-(3-hydroxypyrrolidin-1-yl)but-2-enamideI-104

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(R,E)-4-(3-hydroxypyrrolidin-1-yl)but-2-enoyl in the place of 6 in Step3. MS 485.3 (M+Na).

Example 165 Preparation of(E)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)-4-(1H-pyrazol-1-yl)but-2-enamideI-100

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(E)-4-(1H-imidazol-1-yl)but-2-enoyl chloride in the place of 6 in Step3. MS 466.1 (M+Na).

Example 166 Preparation of(R,E)-N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-4-(3-hydroxypyrrolidin-1-yl)but-2-enamideI-89

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(R,E)-4-(3-hydroxypyrrolidin-1-yl)but-2-enoyl chloride in the place of 7in Step 4. MS 545.3 (M+Na).

Example 167 Preparation of(S,E)-N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-4-(3-hydroxypyrrolidin-1-yl)but-2-enamideI-88

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(S,E)-4-(3-hydroxypyrrolidin-1-yl)but-2-enoyl chloride in the place of 7in Step 4. MS 545.3 (M+Na).

Example 168 Preparation of2-((1H-pyrazol-1-yl)methyl)-N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-85

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using2-((1H-pyrazol-1-yl)methyl)acryloyl chloride in the place of 7 in Step4. MS 526.1 (M+Na).

Example 169 Preparation ofN-(3-(5-fluoro-2-(phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide I-28

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using aniline in the place of4 in Step 2. MS 372.1 (M+Na).

Example 170 Preparation of(E)-4-((3R,5S)-3,5-dimethylpiperazin-1-yl)-N-(3-(5-fluoro-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)but-2-enamideI-119

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(E)-4-((3R,5S)-3,5-dimethylpiperazin-1-yl)but-2-enoyl chloride in theplace of 6 in Step 3. MS 512.3 (M+Na).

Example 171 Preparation of1-(3-(5-methyl-2-(3-aminosulfonylphenylamino)pyrimidin-4-ylamino)phenyl)-3-methylbut-2-en-1-oneI-224

The title compound was prepared according to the schemes, steps andintermediates described in Example 112 using2,4-dichloro-5-methylpyrimine in place of 1 in step-1 and3-aminobenzenesulfonamide in place of 4 in step-2. ¹H NMR (DMSO-d₆) δppm: 1.97 (s, 3H), 2.14 (s, 6H), 6.88 (s, 1H), 7.25-7.30 (m, 4H), 7.47(t, J=7.92 Hz, 1H), 7.62 (d, J=7.72 Hz. 1H), 7.96 (s, 1H), 8.0-8.07 (m,3H), 8.20 (t, J=7.36 Hz, 1H), 8.55 (s, 1H), 9.37 (s, 1H); LCMS: m/e 438(M+1).

Example 172 Preparation ofN-(3-acrylamidophenyl)-N-(5-cyano-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-yl)acrylamideI-171

The title compound was prepared according to the schemes, steps andintermediates described in Example 95 using excess acroyl chloride instep-4. MS m/z: 442.1 (M+H⁺).

Example 173 Preparation ofN-3-(N-methyl-N-(5-fluoro-2-(4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-ylamino)pyrimidin-4-yl)aminophenylacrylamideI-127

The title compound was prepared by treating the product of Example 88with excess formaldehyde and NaBH₃CN (2 equiv.) in acetonitrile andacetic acid (4:1). MS m/z: 435.1 (M+H⁺).

Example 174 Preparation ofN-(3-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)benzyl)acrylamideI-205

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 using2,4-dichloro-5-methylpyrimidine in place of 1 and3-(tert-butoxycarbonylamino)methylaniline in place of 2 in step-1, andaniline in place of 4 on step-2. ¹H-NMR (CDCl₃, 500 MHz): δ 7.91 (s,1H), 7.77 (s, 1H), 7.57 (d, J=9.0 Hz, 2H), 7.41 (d, J=8.0 Hz, 1H),7.36-7.26 (m, 2H), 7.13-6.96 (m, 4H), 6.36-6.25 (m, 2H), 5.97 (dd,J=10.5, 17.0 Hz, 1H), 5.78 (bs, 1H), 5.63 (d, J=10.5 Hz, 1H), 4.51 (d,J=6.0 Hz, 2H), 2.12 (s, 3H). MS: m/e=360 (M⁺+1).

Example 175 Preparation of (E)-3-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino) benzyl but-2-enoate I-246

The title compound was prepared according to the schemes, steps, andintermediates described below.

A) 2, Xanthophos, Pd₂(dba)₃, Cs₂CO₃, CH₃CN, 90° C., 12 hr; B) 4, t-BuOH,90° C., 4 hr; C) 6, TEA, DCM, −30° C., 5 min

Step-1

To a stirred solution of 1 (0.34 g, 2.08 mmol) in acetonitrile (5 mL)were added Cs₂CO₃ (1.09 g, 3.35 mmol), Xanthophos (0.024 g, 0.041 mmol),Pd₂(dba)₃ (38 mg, 0.04 mmol) and 2 (0.5 g, 2.1 mmol) at RT under N₂atmosphere. Argon gas was purged in to the reaction mixture for 1 h andstirred at 90° C. for 12 h. The progress of the reaction was monitoredby TLC. The reaction mixture was filtered through a pad of celite, andthe filtrate was concentrated under reduced pressure. The crude compoundwas purified by silica gel column chromatography to afford 3 (0.56 g,29.16%) as light yellow liquid. ¹H-NMR (CDCl₃, 500 MHz): δ 8.0 (s, 1H),7.55 (s, 1H), 7.51 (d, J=8.0 Hz, 1H), (t, J=7.5 Hz, 1H), (d, J=7.5 Hz,1H), 6.48 (s, 1H), 4.76 (s, 2H), 2.18 (s, 3H), 0.95 (s, 9H), 0.10 (s,6H). MS: m/e=364 [M⁺+1].

Step-2

To a stirred solution of 3 (0.07 g, 0.19 mmol) in t-BuOH (1.5 mL) wasadded aniline (4) (0.018 g, 0.19 mmol) at room temperature. The reactionmixture was heated up to 90° C. and stirred for 4 h at the sametemperature. The progress of the reaction was monitored by TLC. Afterthe completion of starting materials, the volatiles were removed underreduced pressure to give 5 (0.033 g, 55.9%) as light yellow solid.¹H-NMR (DMSO-d₆, 500 MHz): δ 10.45 (s, 1H), 9.82 (s, 1H), 7.91 (s, 1H),7.58-7.01 (m, 9H), 4.50 (s, 2H), 2.16 (s, 3H). MS: m/e=307 [M⁺+1].

Step-3

To a stirred solution of 5 (0.5 g, 1.63 mmol) in DCM (5 mL) was added 6(0.18 g, 1.72 mmol) followed by TEA (0.66 mL, 4.78 mmol) at −30° C.under N₂ atmosphere. The reaction mixture was stirred for 5 minutes at−30° C. and the progress of the reaction was monitored by TLC. After thecompletion of reaction, quenched with water and extracted with DCM (2×50mL). The organic layer was separated, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude material was purified bysilica gel column chromatography to give 50 mg of an isomeric mixture ofthe title compound. This mixture in DCM (2 mL) was treated with DBU(0.02 g, 0.127 mmol) at room temperature. The reaction mixture wasstirred for 2 h at room temperature, quenched with water and extractedwith DCM (2×10 mL). The organic layer was separated, dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford I-246(0.05 g, 10%) as light yellow solid. ¹H-NMR (CDCl₃, 500 MHz): δ 7.87 (s,1H), 7.65 (s, 1H), 7.58-7.51 (m, 3H), 7.34 (t, J=7.5 Hz, 1H), 7.30-7.23(m, 3H), 7.13 (d, J=7.5 Hz, 1H), 7.08-6.96 (m, 2H), 6.40 (s, 1H), 5.87(dd, J=1.5, 15.5 Hz, 1H), 5.16 (s, 2H), 2.13 (s, 3H), 1.87 (dd, J=2.0,7.0 Hz, 3H). ¹³C-NMR (CDCl₃, 125 MHz): δ 166.3, 159.1, 158.4, 155.4,145.3, 139.9, 138.9, 137.0, 128.9, 128.7, 123.2, 122.4, 121.9, 121.2,121.0, 119.3, 105.2, 65.7, 17.9, 13.2. MS: m/e=375 [M⁺+1].

Example 176 Preparation of (E)-4-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino) benzyl but-2-enoate I-60

The title compound was prepared according to the schemes, steps andintermediates described in Example 175 using4-((tert-butyldimethylsilyloxy) methyl) aniline in place of 2 in step-1.¹H-NMR (CDCl₃, 500 MHz): δ 8.19 (bs, 1H), 7.81 (s, 1H), 7.56 (d, J=8.5Hz, 2H), 7.53 (d, J=7.5 Hz, 2H), 7.42-7.36 (m, 3H), 7.28-7.22 (m, 1H),7.08-6.98 (m, 2H), 6.54 (s, 1H), 5.89 (dd, J=12.5, 14.0 Hz, 1H), 5.17(s, 2H), 2.15 (s, 3H), 1.89 (dd, J=1.5, 7.0 Hz, 3H). MS: m/e=375 [M⁺+1].

Example 177 Preparation ofN-methyl-N-(3-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)benzyl)acrylamideI-220

The title compound was prepared according to the schemes, steps, andintermediates described below.

A) 2, Xanthophos, Pd₂(dba)₃, Cs₂CO₃, CH₃CN, 100° C., 12 hr; B) 4,t-BuOH, 90° C., 4 hr; C) 10 N HCl, DCM, rt, 30 min: D) 7, TEA, DCM, −10°C., 10 min.

Step-1

To a stirred solution of 1 (2.6 g, 15.7 mmol) in acetonitrile (26.7 mL)was added 2 (2.67 g, 11.3 mmol), Pd₂ (dba)₃ (0.31 g, 0.33 mmol),Xanthophos (0.52 g, 0.89 mmol) and Cs₂CO₃ (6.6 g, 20.0 mmol). Thereaction mixture was then degassed by purging argon for 1 h and furtherheated to 100° C. for 12 h. After the completion of the reaction(monitored by TLC), the reaction mixture was filtered through celite bedand the filtrate was concentrated under reduced pressure. The resultingcrude material was purified by column chromatography (60-120 mesh silicagel; 20% ethyl acetate/Hexane) to afford 3 (2.32 g, 56.71%) as lightbrown solid. ¹H-NMR (CDCl₃, 500 MHz): □ 8.01 (s, 1H), 7.56 (d, J=7.5 Hz,1H), 7.43 (s, 1H), 7.35 (t, J=7.0 Hz, 1H), 7.05 (s, 1H), 6.80 (bs, 1H),4.45 (s, 2H), 2.87 (s, 3H), 2.30 (s, 3H), 1.48 (s, 9H).

Step-2

To a stirred solution of 3 (2.32 g, 6.0 mmol) in t-BuOH (11.6 mL) wasadded 4 (0.65 g, 6.9 mmol) at RT and the reaction mixture was furtherheated at reflux for 48 h. The progress of the reaction was monitored byTLC. After the completion of the reaction, t-BuOH was concentrated underreduced pressure to dryness to give 5 (2.3 g, 85.82%) as light yellowsolid. ¹H-NMR (DMSO-d₆, 500 MHz): δ 10.32 (s, 1H), 9.78 (s, 1H), 7.91(s, 1H), 7.50 (d, J 8.0 Hz, 1H), 7.45-7.30 (m, 4H), 7.24 (t, J=7.0 Hz,2H), 7.15-7.06 (m, 2H), 4.36 (s, 2H), 2.72 (s, 3H), 2.17 (s, 3H), 1.41,1.34 (two s, 9H). MS: m/e=420 (M⁺+1).

Step-3

To a stirred solution of 5 (0.05 mg, 0.11 mmol) in DCM (5 mL) was added37% HCl (1.0 mL) and stirred at RT for 30 min. After the completion ofthe reaction (monitored by TLC), volatiles were removed under reducedpressure. The aqueous layer was cooled to 0° C., basified up to pH˜8-9with 10% NaOH solution and extracted with DCM (50 mL). The organicportion was separated, washed with water, brine, dried over anhydrousNa₂SO₄ and evaporated under reduced pressure to afford 6 (0.015 g,48.36%) as light yellow solid. ¹H-NMR (CDCl₃, 500 MHz): δ 7.95-7.85 (m,2H), 7.68-7.60 (m, 3H), 7.42 (d, J=8.0 Hz, 1H), 7.32-7.20 (m, 4H), 7.05(d, J=7.5 Hz, 1H), 7.00-6.95 (m, 1H), 6.40 (s, 1H), 3.84 (s, 2H), 2.48(s, 3H), 2.06 (s, 3H). MS: m/e=320 (M⁺+1).

Step-4

To a stirred solution of 6 (0.3 g, 0.94 mmol) in DCM (12 mL) was addedTEA (0.10 g, 0.99 mmol) and 7 (0.08 g, 0.88 mmol) dropwise over a periodof 5 min at −10° C. under inert atmosphere. The reaction mixture wasthen stirred at −10° C. for 5-10 min. After the completion of thereaction (monitored by TLC), the reaction mixture was quenched with coldwater (5 mL) and extracted with DCM (2×50 mL). The DCM layer was washedwith water, brine, dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The crude material was purified by columnchromatography (60-120 mesh silica gel; 30% Ethyl acetate/Hexane) toafford I-220 (0.15 g, 42.85%) as off white solid. ¹H-NMR (DMSO-d₆, 500MHz, at 80° C.): δ 8.48 (bs, 1H), 8.07 (s, 1H), 7.88 (s, 1H), 7.69-7.58(m, 4H), 7.28 (t, J=8.0 Hz, 1H), 7.16 (t, J=8.0 Hz, 2H), 6.91-6.85 (m,2H), 6.75 (dd, J=2.5, 15.3 Hz, 1H), 6.13 (d, J=15.0 Hz, 1H), 5.66 (d,J=7.5 Hz, 1H), 4.60 (s, 2H), 2.95 (s, 3H), 2.12 (s, 3H). MS: m/e=374(M⁺+1).

Example 178 Preparation ofN-methyl-N-(4-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)benzyl)acrylamideI-219

The title compound was prepared according to the schemes, steps andintermediates described in Example 177 usingtert-butyl-4-aminobenzyl(methyl)-carbamate in place of 2 in step-1. ¹HNMR (DMSO-d₆, 500 MHz at 80° C.): δ 8.55 (s, 1H), 8.03 (s, 1H), 7.86 (s,1H), 7.65 (d, J=8.0 Hz, 2H), 7.62 (d, J=8.0 Hz, 2H), 7.20-7.13 (m, 4H),6.86-6.75 (m, 2H), 6.14 (dd, J=2.5, 17.0 Hz, 1H), 5.67 (d, J=15.0 Hz,1H), 4.58 (s, 2H), 2.96 (s, 3H), 2.10 (s, 3H). MS: m/e=374 (M⁺+1).

Example 179 Preparation ofN-(5-(5-acetyl-4-(4-acryloyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-ylamino)pyrimidin-2-ylamino)pyridin-2-yl)-2,2,2-trifluoro-N-methylacetamideI-142

The title compound was prepared according to the schemes, steps andintermediates described in Example 42 using5-amino-2(2,2,2-trifluoroacetamido)pyridine in place of 9 in step-5.LC-MS: m/z 542.2 (ES+), 540.2.2 (ES−).

Example 180 Preparation of1-(6-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)indolin-1-yl)prop-2-en-1-oneI-94

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 using N-Boc-6-aminoindoline inplace of 2 in step-1. LC-MS: m/z 450.1 (ES+), 448.1 (ES−).

Example 181 Preparation ofN-(3-(5-fluoro-2-(6-(2-methoxyethoxy)pyridine-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-103

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 by using3-amino-6-(2-methoxyethoxy)pyridine in place of 4 in Step-2. ¹H NMR(CDCl₃+trace of DMSO-d₆) δ ppm: 3.44 (s, 3H), 3.75 (t, J=4.4 Hz, 2H),4.43 (t, J=4.4 Hz, 2H), 5.81 (dd, J=1.8 & 9.6 Hz, 1H), 6.45 (m, 1H),6.80 (m, 3H), 7.17 (m, 1H), 7.29 (m, 1H), 7.43 (m, 1H), 7.49 (m, 1H),7.60 (m, 1H), 7.80 (dd, J=2.8 & 9.2 Hz, 1H), 7.94 (d, J=3.1 Hz, 1H),8.14 (s, 1H), 8.32 (d, J=2.9 Hz, 1H); LCMS: m/e 425.1 (M+1).

Example 182 Preparation ofN-(3-(5-fluoro-2-(4-(3-methylsulfonylpropoxy)phenyl)aminopyrimidin-4-ylamino)phenyl)acrylamideI-97

The title compound was prepared as a TFA salt according to the schemes,steps and intermediates described in Example 20 by using4-(3-methylsulfonylpropoxy)aniline in place of 4 in Step-2. ¹H NMR(CDCl₃+trace of DMSO-d₆) δ ppm: 1.95 (m, 2H), 2.67 (s, 3H), 2.98 (m,5H), 3.74 (t, J=6.0 Hz, 2H), 5.45 (dd, J=4.1 & 7.3 Hz, 1H), 6.07 (m,2H), 6.48 (d, J=8.2 Hz, 1H), 6.77 (m, 4H), 7.09 (d, J=7.4 Hz, 1H), 7.51(d, J=4.1 Hz, 1H), 7.70 (br, 1H); LCMS: m/e 486.1 (M+1).

Example 183 Preparation ofN-(3-(5-fluoro-2-(6-(trideutereomethoxy)pyridine-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-95

The title compound was prepared as a TFA salt according to the schemes,steps and intermediates described in Example 20 by using6-(trideuteriomethoxy)pyridin-3-amine in place of 4 in Step-2. ¹H NMR(CDCl₃+trace of DMSO-d₆) δ ppm: 5.78 (dd, J=3.7 & 7.8 Hz, 1H), 6.40 (m,2H), 6.71 (d, J=8.7 Hz, 1H), 7.3 (m, 3H), 7.75 (dd, J=2.7 & 8.7 Hz, 1H),7.82 (d, J=4.6 Hz, 1H), 7.95 (s, 1H), 8.33 (d, J=2.3 Hz, 1H); LCMS: m/e384.1 (M+1).

The intermediate 6-(trideutratedmethoxy)pyridin-3-amine was prepared bythe scheme shown below.

A) NaH, CD₃OD, rt; BH₃NMe₃, Pd(OH)₂

Step 1

To NaH (60%, 0.30 g) in 5 mL of CD₃OD at 0° C. was added2-chloro-5-nitropyridine (1.0 g). The mixture was stirred at rtovernight. To this mixture were added BH₃.NMe₃ (550 mg) and Pd(OH)₂ (100mg). The resulting mixture was refluxed for 2 h. After cooling down, themixture was concentrated and purified using silica gel chromatography togive the desired 6-(trideuteratedmethoxy)pyridin-3-amine (130 mg). ¹HNMR (CDCl₃) δ ppm: 3.30 (br, 2H), 6.60 (d, J=8.7 Hz, 1H), 7.03 (dd,J=3.2 & 8.7 Hz, 1H), 7.66 (d, J=3.2 Hz, 1H).

Example 184 Preparation ofN-(3-(5-fluoro-2(3,4,5-trimethoxyphenylamino)pyrimidin-4-yloxy)phenyl)acrylamideI-148

The title compound was prepared according to the schemes, steps andintermediates described in Example 98 by using 3,4,5-trimethoxyanilinein place of 4 in Step-2. MS: m/e 441 [M+1].

Example 185 Preparation of3-methyl-1-(3-(5-methyl-2-(phenylamino)pyrimidin-4-ylamino)phenyl)but-2-en-1-oneI-232

The title compound was prepared according to the schemes, steps andintermediates described in Example 112 using2,4-dichloro-5-methylpyrimidine in place of 1 in step-1 and aniline inplace of 4 in step-2. ¹H NMR (CDCl₃) δ ppm: 1.97 (s, 3H), 2.15 (s, 3H),2.22 (s, 3H), 6.47 (s, 1H), 6.71 (s, 1H), 6.97 (t, J=9.8 Hz, 1H), 7.17(s, 1H), 7.24 (t, J=10.36 Hz, 1H), 7.27 (s, 1H), 7.44 (t, J=10.64 Hz,1H), 7.53 (d, J=10.48 Hz, 2H), 7.68 (d, J=10.28 Hz, 1H), 7.94 (d, J=10Hz, 1H), 7.98 (s, 1H); LCMS: m/e 359 (M+1).

Example 186 Preparation of1-(3-(5-methyl-2-phenylamino)pyrimidin-4-ylamino(piperidin-1-yl)prop-2-en-oneI-27

The title compound was prepared according to the schemes, steps andintermediates described in Example 1 using1-tert-butoxycarbonyl-3-aminopiperidine in place of 1 in step-1. ¹H NMR(DMSO-d₆) δ ppm: 1.30-1.50 (m, 1H), 1.55-1.75 (m, 1H), 1.75-1.90 (m,1H), 1.92 (s, 3H), 1.95-2.05 (m, 1H), 2.75-3.31 (m, 2H), 3.99-4.09 (m,2H), 4.10-4.15 & 4.40-4.47 (m, 1H), 5.49 & 5.70 (d, J=10.8 Hz & d, J=9.2Hz respectively, together 1H), 6.02 & 6.13 (d, J=17.6 Hz & d, J=16.8 Hzrespectively, together 1H), 6.25-6.40 (m, 1H), 6.63 & 6.80-6.90 (dd,J=10.8, 16.8 Hz & m respectively, together 1H), 6.75-6.85 (m, 1H), 7.15(t, J=8 Hz, 2H), 7.69 (bs, 3H), 8.81 (s, 1H); LCMS: m/e 337.8 (M+1).

Example 187 Preparation of3-(4-(2-acryloyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-5-methylpyrimidin-2-ylamino)benzenesulfonamideI-40

The title compound was prepared according to the schemes, steps andintermediates described in Example 1 using6-amino-2-tert-butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline in place of1 in step-1. ¹H NMR (DMSO-d₆) δ ppm: 2.10 (s, 3H), 2.80-2.83 (m, 2H),3.75-3.90 (m, 2H), 4.66 (s, 1H), 4.76 (s, 1H), 5.71-5.74 (m, 1H), 6.16(dd, J=2.32 & 16.76 Hz, 1H), 6.87-6.91 (m, 1H), 7.13-7.18 (m, 1H),7.25-7.31 (m, 4H), 7.53-7.57 (m, 2H), 7.90 (s, 1H), 8.05 (s, 2H), 8.28(s, 1H), 9.31 (s, 1H); LCMS: m/e 464.8 (M+1).

Example 188 Preparation of(S)—N-(3-(5-fluoro-2-(tetrahydrofuran-3-yloxy)pyridine-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-54

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 using(S)-3-amino-6-(tetrahydrofuran-3-yloxy)pyridine in place of 4 in step-2.MS: m/e=437 [M+1].

Example 189 Preparation ofN-(3-(5-trifluoromethyl-2-(phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-245

The title compound was prepared according to the schemes, steps, andintermediates described below.

A) 2, ZnCl₂, DCE, t-BuOH (1:1), 0° C., 30 min; B) 4, DMF, DIEPA, 70° C.,16 hr; C) TFA, DCM, rt, 1 hr; D) 7, TEA, DCM.

Step-1

To a cold (0° C.) solution of 1 (2 g, 9.2 mmol) in 80 mL of a 1:1mixture of tBuOH/DCE was added zinc chloride (11 mL of a 1 M solution inether, 1.2 eq). After one hour, 2 (0.858 g, 9.2 mmol) was added followedby dropwise addition of triethylamine (1.03 g; 1.1 eq) in 10 mL ofDCE/t-BuOH. After stirring for 30 minutes, the solvents were removedunder reduced pressure and the residue was dissolved in ethyl acetate(50 mL) and washed with brine (10 mL). The organic layer was dried oversodium sulphate, filtered and concentrated in vacuo. The desired product3 was obtained as a white solid following recrystalization fromEtOAc/Hexane (1:9), (2 g, 80%).

Step-2

To a solution of 3 (0.5 g, 1.82 mmol) and 4 (0.38 g, 1.83 mmol) in DMF(10 mL) was added DIPEA (0.283 g, 2.192 mmol) and the mixture was heatedto 60° C. under an argon atmosphere for 16 h. The solvent was distilledoff and the residue was dissolved in ethyl acetate (50 mL) and washedwith brine (10 mL). The organic layer was dried over sodium sulphate,filtered and concentrated in vacuo. The crude mixture was purified byflash column chromatography (eluent: EtOAc/hexane 1:1) to afford 5 as awhite solid (0.48 g, 60%).

Step-3

To a solution of 6 (0.25 g, 0.63 mmol) in CH₂Cl₂ (10 mL) was addedtrifluoroacetic acid (2 mL) and the mixture was stirred at roomtemperature for 1 hour. Solvents were removed under reduced pressure andthe residue was dissolved in CH₂Cl₂, washed with 10% aqueous NaHCO₃solution, dried (Na₂SO₄), filtered, and evaporated under reducedpressure to provide the free amine as white solid.

Step-4

To a stirred solution of 6 (0.2 g) in DCM (20 mL) under argon atmospherecooled to −40° C. was added triethylamine followed by dropwise additionof 7 (0.069 g, 0.686 mmol). The resulting mixture was stirred at −40° C.for 10 min. The reaction mixture was diluted with DCM (50 mL) and washedwith brine (10 mL). The organic layer was dried over sodium sulfate,filtered, and evaporated under reduced pressure. The residue waspurified by flash chromatography on silica gel using (MeOH-EtOAc 5:95)as eluent to provide the target compound I-245. ¹H NMR (200 MHz, CD₃OD)δ 8.25 (s, 1H), 7.80 (s, 1H), 7.60-7.05 (m, 7H), 6.90 (m, 1H), 6.35 (m,2H), 5.75 (dd, J=8.0, 2.0 Hz, 1H).

Example 190 Preparation ofN-(3-(5-trifluoromethyl-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-242

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-methoxyaniline in placeof 2 in step-1. ¹H NMR (200 MHz, CD₃OD) δ 8.31 (s, 1H), 7.84 (s, 1H),7.59 (m, 1H), 7.37-7.09 (m, 5H) 6.53 (m, 1H), 6.41 (m, 2H), 5.79 (dd,J=8.0, 2.0, Hz, 1H), 3.66 (s, 3H).

Example 191 Preparation ofN-(4-(5-trifluoromethyl-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-236

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-methoxyaniline in placeof 2 in step-1 and 4-amino-N-tert-butoxycarbonylaniline in place of 4 instep-2. ¹H NMR (200 MHz, CD₃OD) δ 8.27 (s, 1H), 7.70 (d, J=6.0 Hz) 1H),7.46 (d, J=6.0 Hz, 1H), 7.09 (brs, 1H), 7.07 (m, 2H) 6.51 (m, 1H), 6.44(m, 2H), 5.80 (dd, J=8.0, 2.0 Hz, 1H), 3.56 (s, 3H).

Example 192 Preparation ofN-(4-(5-trifluoromethyl-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)phenyl)methylacrylamideI-235

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-methoxyaniline in placeof 2 in step-1 and 4-aminophenylmethyl-N-tert-butoxycarbonylamine inplace of 4 in step-2. ¹H NMR (200 MHz, CD₃OD) δ 8.30 (s, 1H), 7.49 (d,J=8.0 Hz, 1H), 7.37 (d, J=8.0 Hz, 1H), 7.10 (m, 3H), 6.60 (m, 1H) 6.34(m, 2H), 5.75 (dd, J=8.0, 2.0 Hz, 1H), 4.51 (s, 2H), 3.68 (s, 3H).

Preparation ofN-(4-chloro-3-(5-trifluoromethyl-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-227

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-methoxyaniline in placeof 2 in step-1 and N-tert-butoxycarbony-3-amino-6-chloroaniline in placeof 4 in step-2. ¹H NMR (200 MHz, CD₃OD) δ 8.33 (s, 1H), δ 8.08 (s, 1H),7.45 (m, 2H), 7.21-7.07 (m, 3H), 6.60-6.36 (m, 3H), 5.84 (dd, J=8.0, 2.0Hz, 1H), 3.71 (s, 3H).

Example 194 Preparation ofN-(3-(5-trifluoromethyl-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)phenyl)methylacrylamideI-226

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-methoxyaniline in placeof 2 in step-1 and 3-aminophenylmethyl-N-tert-butoxycarbonylamine inplace of 4 in step-2. ¹H NMR (200 MHz, CD₃OD) δ 8.31 (s, 1H), 7.71-7.33(m, 3H), 7.21-7.08 (m, 4H), 6.57 (m, 1H), 6.26 (d, J=4 Hz, 2H), 5.69(dd, J=8.0, 2.0 Hz, 1H), 4.47 (s, 2H), 3.67 (s, 3H).

Example 195 Preparation ofN-(4-(5-trifluoromethyl-2-(6-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-218

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-amino-6-methoxypyridinein place of 2 in step-1. ¹H NMR (200 MHz, CD₃OD) δ 8.21 (s, 1H),7.90-7.78 (m, 3H), 7.48 (m, 2H), 7.30 (m, 2H), 6.40 (m, 2H), 5.75 (dd,J=8.0, 2.0 Hz, 1H), 3.81 (s, 3H).

Example 196 Preparation ofN-(4-(5-trifluoromethyl-2-(5-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-214

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-amino-5-methoxypyridinein place of 2 in step-1 and 4-amino-N-tert-butoxycarbonylaniline inplace of 4 in step-2. MS: m/e=431 [M+1].

Example 197 Preparation of1-(3-(5-trifluoromethyl-2-(3-methoxyphenylamino)pyrimidin-4-ylamino)phenyl)-3-methyl-but-2-en-1-oneI-225

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-methoxyaniline in placeof 2 in step-1 and 1-(3-aminophenyl)-3-methylbut-2-en-1-one in place of4 in step-2. ¹H NMR (200 MHz, CDCl₃) δ 8.33 (s, 1H), δ 8.38 (s, 1H),7.99-7.77 (m, 4H), 7.50 (m, 2H), 7.20-7.01 (m, 4H), 6.68-6.60 (m, 2H),3.68 (s, 3H), 2.25 (s, 3H), 1.99 (s, 3H).

1-(3-Aminophenyl)-3-methylbut-2-en-1-one was prepared according to thescheme, steps, and intermediates described below.

A) Boc₂O, NEt₃, DMAP, DCM; B) NHMe(OMe)-HCl, TBTU, DCM, 0° C. to rt; C)4, THF, 0° C. to rt; D) TFA, DCM.

Step-1

Di-tert-butyldicarbonate (6.54 g, 30 mmol, 1.5 eq.) was added to asolution of 1 (2.70 g, 20 mmol) in CH₂Cl₂ (100 mL) containing Et₃N (3.4mL, 24 mmol, 1.2 eq.) and DMAP (122 mg, 1.0 mmol, 5 mol %). The mixturewas stirred overnight under a CaCl₂ drying tube. The solvents wereevaporated and the residue was partitioned between ether (50 mL) andwater (50 mL). The aqueous phase was extracted with ether then acidifiedto pH 3 with 1N HCl and extracted with EtOAc (2×50 mL). The combinedorganic layers were washed with water and brine and dried over NaSO₄.Concentration afforded the crude product which was recrystallized fromEtOAc/hexanes to give 2 (2.92 g, 62%).

Step-2

To a mixture of 2 (1.5 g, 6.33 mmol), N-methoxy-N-methylaminehydrochloride (614 mg, 6.33 mmol), and TBTU (2.05 g, 6.33 mmol) in DCM(30 mL) at 0° C. was added NEt3 (2.7 mL, 19 mmol, 3 eq.). The mixturewas stirred for 30 min at 0° C. then at room temperature for 2 hwhereupon HPLC analysis indicated the reaction to be complete. Thereaction mixture was poured into 150 mL of cold water and the productprecipitated as a white solid which was collected and washed with coldwater. The product was dried in a vacuum oven overnight to give 3 (1.34g, 75%) as a white solid.

Step-3

To a solution of 3 (546 mg, 1.95 mmol) in THF (3 mL) at 0° C. under Arwas added dropwise 4 (0.5 M in THF, 9.75 mL, 4.9 mmol, 2.5 eq.). Thereaction mixture was stirred at 0° C. for 30 min then the cooling bathwas removed and the reaction was stirred at rt for 2 h. The reactionmixture was cooled to 0° C. and quenched with 5% citric acid solution.After dilution with water (10 mL) the aqueous phase was extracted withether (2×15 mL) and the combined organic layers were washed with waterand brine and dried over NaSO₄. Concentration afforded 5 (82%) as ayellow solid which was sufficiently pure to be used directly in the nextstep.

Step-4

A sample of 400 mg of 5 was treated with 5 mL 1:3 CH₂Cl₂:trifluoroaceticacid and the resulting solution stirred at room temperature for 15minutes. The solvents were removed in vacuo and the residue redissolvedin CH₂Cl₂ and re-evaporated three times. The residue was again taken upin CH₂Cl₂ and the solution washed with saturated sodium bicarbonatesolution. The CH₂Cl₂ layer was dried over sodium sulphate, filtered andevaporated to afford 6 as a white solid that was used directly in thenext reaction.

Example 198 Preparation of1-(3-(2-(3-Methoxy-phenylamino)-5-trifluoromethyl-pyrimidin-4-ylamino)-cyclohexyl)-3-methyl-but-2-en-1-oneI-213

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-methoxyaniline in placeof 2 in step-1 and(d,l)-cis-1-(3-Amino-cyclohexyl)-3-methyl-but-2-en-1-one in place of 4in step-2. ¹H NMR (200 MHz, CDCl₃) δ 8.07 (s, 1H), 7.33 (s, 1H),7.19-7.0 (m, 3H), 6.54 (d, J=2.7 Hz, 1H), 6.02 (s, 1H), 4.04 (m, 1H),3.75 (s, 3H), 2.50 (m, 1H), 2.10 (m, 1H), 1.89-1.15 (m, 14H).

(D,L)-cis-1-(3-Amino-cyclohexyl)-3-methyl-but-2-en-1-one was preparedaccording to the scheme, steps, and intermediates described below.

A) Boc₂O, Na₂CO₃, acetone, H₂O; B) NHMe(OMe)-HCl, TBTU, DCM, 0° C. tort; C) 4, THF, 0° C. to rt; D) TFA, DCM.

Step-1

To a stirred solution of (±)-1 (4.05 g, 28.2 mmol) in water (150 mL)containing Na₂CO₃ (3.0 g, 28.2 mmol) and acetone (100 mL) was addedBOC₂O (7.4 g, 33.8 mmol, 1.2 eq) and the mixture was stirred at 25° C.overnight. The acetone was stripped and the aqueous layer was extractedwith ether (2×). The aqueous layer was acidified to pH 3 and theprecipitated product was collected and washed with water. The productwas dried in a vacuum oven overnight to give 2 (5.90 g, 85%) as a whitesolid.

Step-2

To a stirred solution of 2 (2.45 g, 10.1 mmol), TBTU (3.44 g, 10.6 mmol,1.05 eq) and N-methyl-N-methoxyamine hydrochloride (1.03 g, 10.6 mmol,1.05 eq) in CH₂Cl₂ (40 mL) at 0° C. was added triethylamine (4.25 mL,30.3 mmol, 3 eq). The mixture was stirred at 0° C. for 20 min and thebath was removed and stirring was continued for 3 h at 25° C. Afterquenching with water, the CH₂Cl₂ was stripped and the residue waspartitioned between ether and water. The aqueous phase was extractedwith ether and the combined organic layers were washed with water andbrine and dried over MgSO₄. Evaporation of the solvents gave 3 (2.37 g,82%) as a white solid.

Step-3

To a solution of 3 (1.23 g, 4.32 mmol) in THF (20 mL) at 0° C. underargon was added dropwise 4 (27 mL, 0.5 M in THF, 10.8 mmol, 2.5 eq).After the addition was complete the mixture was stirred at 0° C. for 30min, then at rt for 1 h. The reaction mixture was cooled to 0° C. thenquenched with 5% citric acid solution (5 mL). After dilution with waterthe mixture was extracted with ether (2×) and the combined organiclayers were washed with water and brine and dried over NaSO₄.Evaporation left an orange residue which was chromatographed on silicagel eluting with 20% EtOAc in hexanes to give 5 (600 mg, 56%) as a lightyellow solid.

Step-4

A sample of 500 mg of 5 was treated with 6 mL 1:3 CH₂Cl₂:trifluoroaceticacid and the resulting solution stirred at room temperature for 15minutes. The solvents were removed in vacuo and the residue redissolvedin CH₂Cl₂ and re-evaporated three times. The residue was again taken upin CH₂Cl₂ and the solution washed with saturated sodium bicarbonatesolution. The CH₂Cl₂ layer was dried over sodium sulphate, filtered andevaporated to afford 6 as a white solid that was used directly withoutpurification.

Example 199 Preparation of1-(5-(5-trifluoromethyl-2-(3-methoxyphenylamino)pyrimidin-4-yl)amino-1,3-dihydroisoindol-2-yl)2-propen-1-oneI-132

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using 3-methoxyaniline in placeof 2 in step-1 and 2-(N-tert-butoxycarbonyl)-5-aminoisoindoline in placeof 4 in step-2. MS m/e=456 [M+1].

Example 200 Preparation of3-(2-(2-acryloylisoindolin-5-ylamino)-5-fluoropyrimidin-4-ylamino)benzonitrileI-106

The title compound was prepared according to the schemes, steps andintermediates described in Example 2 using5-fluoro-2,4-dichloropyrimidine in place of 1 and 3-aminobenzonitrile inplace of 2 in step-1 and 2-(tert-butoxycarbonyl-5-aminoisoindoline inplace of 4 in step-2. LC/MS (RT=2.82/(M+H)) 401.1

Example 201 Preparation ofN-(3-(5-fluoro-4-((6-(trifluoromethyl)pyridin-3-yl)methylamino)pyrimidin-2-ylamino)phenyl)acrylamideI-53

The title compound was prepared according to the schemes, steps andintermediates described in Example 2 using5-fluoro-2,4-dichloropyrimidine in place of 1 and3-aminomethyl-6-trifluoromethylpyridine in place of 2 in step-1. LC/MS(RT=2.805/(M+H)) 433.0

Example 202 Preparation ofN-(3-(4-((2,3-dihydrobenzofuran-5-yl)methylamino)-5-fluoropyrimidin-2-ylamino)phenyl)acrylamideI-6

The title compound was prepared according to the schemes, steps andintermediates described in Example 2 using5-fluoro-2,4-dichloropyrimidine in place of 1 and3-aminomethyl-2,3-dihydrobenzofuran in place of 2 in step-1. LC/MS(RT=2.815/(M+H)) 406.2

Example 203 Preparation ofN-(3-(5-fluoro-2-(4-methoxybenzylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-241

The title compound was prepared according to the schemes, steps andintermediates described in Example 20 using 4-methoxybenzylamine inplace of 4 in step-2. LC/MS (RT=2.801/(M+H)) 394.2

Example 204 Preparation ofN¹-(3-(3-(4-(3-acrylamidophenylamino)-5-methylpyrimidin-2-ylamino)phenoxy)propyl)-N⁵-(15-oxo-19-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramideI-215

The title compound was prepared according to the schemes steps andintermediates described below.

A) TFA, DCM; B)N-Biotinyl-NH-(PEG)₂-COOH-DIPEA, HOBt, EDC, NMM, DMF.

Step-1

I-45 (97 mg, 0.19 mmol; synthesis of I-45 provided in Example 62) wasdissolved in DCM (10 mL). Trifluoroacetic acid (200 μL) was added andallow to stir at rt for 24 hr. The solvent was removed via rotaryevaporation to give a tan-brown foam (130 mg) which was used withoutpurification in the next reaction. LC/MS (RT=2.63/(MH+) 419.2)

Step-2

1 (80 mg, 0.15 mmoL) was dissolved in DMF (2 mL). To the mixture wasadded N-Biotinyl-NH-(PEG)₂-COOH-DIPEA (114 mg, 0.16 mmol) and HOBt (25mg, 0.16 mmoL (89%)), and the mixture was cooled in an ice-water bath.EDC (32 mg, 0.16 mmoL) was added, followed by N-methylmorpholine (50 μL,0.45 mmoL). The mixture was allowed to warm to room temperature andcontinue to stir for 30 min. Direct purification by flash chromatographyusing 10% gradient of MeOH in DCM gave 40 mg of I-215 as a yellow film.LC/MS (RT=2.654/(MH+) 961.3).

Example 205 Preparation ofN-(3-(3-(4-(3-acrylamidophenylamino)-5-methylpyrimidin-2-ylamino)phenoxy)propyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamideI-237

The title compound was prepared according to the schemes, steps andintermediates described in Example 204 using D-(+)-biotin in place ofN-Biotinyl-NH-(PEG)₂-COOH-DIPEA in step-2. LC/MS (RT=2.686/(M+H)) 645.2

Example 206 Preparation of(R)—N-(3-(5-fluoro-2-(3-fluoro-4-(tetrahydrofuran-3-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-316

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(R)-3-fluoro-4-(tetrahydrofuran-3-yloxyaniline in place of 4 in step 2.¹H NMR (DMSO-d₆) δ ppm: 1.85-2.00 (m, 1H), 2.20 (m, 1H), 3.70-3.90 (m,4H), 4.90 (s, 1H), 5.73 (dd, J=1.56 & 10.04 Hz, 1H), 6.23 (dd, J=1.76 &17.00 Hz, 1H), 6.44 (dd, J=10.08 & 16.88 Hz, 1H), 7.28 (t, J=8.04 Hz,1H), 7.40-7.47 (m, 2H), 7.67-7.71 (m, 2H), 7.68 (dd, J=1.96 & 14.08 Hz,1H), 7.92 (s, 1H), 8.1 (d, J=3.64 Hz, 1H), 9.21 (s, 1H), 9.44 (s, 1H),10.12 (s, 1H); LCMS: m/e 452.0 (M−1).

Example 207 Preparation of1-(4-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-3-methylbut-2-en-1-oneI-325

The title compound was prepared according to the schemes, steps andintermediates described in Example 112, by using methyl 4-aminobenzoatein place of 2 in step 1 and 3-fluoro-4-(2-methoxyethoxy)aniline in placeof 4 in step 2. ¹H NMR (DMSO-d₆) δ ppm: 2.01 (s, 3H), 2.15 (d, J=0.72Hz, 3H), 3.31 (s, 3H), 3.66 (dd, J=3.64 & 4.56 Hz, 2H), 4.11 (dd, J=4.44& 6.12 Hz, 2H), 6.93 (s, 1H), 7.08 (t, J=9.44 Hz, 1H), 7.27 (d, J=8.88Hz, 1H), 7.74 (dd, J=2.44 & 14.24 Hz, 1H), 7.93 (d, J=8.96 Hz, 2H), 7.98(d, J=8.92 Hz, 2H), 8.20 (d, J=3.64 Hz, 1H), 9.35 (s, 1H), 9.73 (s, 1H);LCMS: m/e 455 (M+1).

Example 208 Preparation of1-(3-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-3-methylbut-2-en-1-oneI-323

The title compound was prepared according to the schemes, steps andintermediates described in Example 112, by using2-(3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2. ¹H NMR(DMSO-d₆) δ ppm: 1.95 (s, 3H), 2.14 (s, 3H), 3.31 (s, 3H), 3.63 (t, J4.64 Hz, 2H), 4.06 (t, J=4.36 Hz, 2H), 6.85 (bs, 1H), 6.97 (t, J=9.52Hz, 1H), 7.26 (bd, J=8.32 Hz, 1H), 7.48 (t, J=7.92 Hz, 1H), 7.62-7.67(m, 2H), 8.08 (bd, J=7.04 Hz, 1H), 8.15-8.16 (m, 2H), 9.29 (s, 1H), 9.58(s, 1H); LCMS: m/e 455 (M+1).

Example 209 Preparation of1-(4-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-2-methylprop-2-en-1-oneI-324

The title compound was prepared according to the schemes, steps andintermediates described in Example 112, by using methyl 4-aminobenzoatein place of 2 in step 1, 3-fluoro-4-(2-methoxyethoxy)aniline in place of4 in step 2 and isopropenylmagnesium bromide in place of 8 in step 5. ¹HNMR (DMSO-d₆) δ ppm: 2.0 (s, 3H), 3.32 (s, 3H), 3.66 (t, J=4.36 Hz, 2H),4.11 (t, J=4.44 Hz, 2H), 5.55 (s, 1H), 5.94 (s, 1H), 7.07 (t, J=9.32 Hz,1H), 7.26 (t, J=9.4 Hz, 1H), 7.72-7.76 (m, 3H), 7.99 (d, J=8.44 Hz, 2H),8.21 (d, J=3.56 Hz, 1H), 9.38 (s, 1H), 9.75 (s, 1H); LCMS: m/e 441.2(M+1).

Example 210 Preparation of1-(4-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-3-methylbut-3-en-2-oneI-329

The title compound was prepared according to the schemes, steps andintermediates described in Example 112, by using ethyl4-aminophenylacetate in place of 2 in step 1,3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2 andisopropenylmagnesium bromide in place of 8 in step 5. ¹H NMR (DMSO-d₆) δppm: 1.79 (s, 3H), 3.30 (s, 3H), 3.62-3.65 (m, 2H), 4.06 (s, 2H),4.08-4.10 (m, 2H), 5.95 (d, J=1 Hz, 1H), 6.27 (s, 1H), 7.01 (t, J=9.44Hz, 1H), 7.15 (d, J=8.52 Hz, 2H), 7.28 (d, J=8.88 Hz, 1H), 7.64-7.70 (m,3H), 8.08 (d, J=3.72 Hz, 1H), 9.19 (s, 1H), 9.33 (s, 1H); LCMS: m/e455.3 (M+1).

Example 211 Preparation of1-(4-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-4-methylpent-3-en-2-oneI-331

The title compound was prepared according to the schemes, steps andintermediates described in Example 112, by using ethyl4-aminophenylacetate in place of 2 in step 1,3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2. ¹H NMR(DMSO-d₆) δ ppm: 1.84 (d, J=1 Hz, 3H), 2.05 (d, J=0.92 Hz, 3H), 3.30 (s,3H), 3.62-3.64 (m, 2H), 3.68 (s, 2H), 4.07-4.09 (m, 2H), 6.21 (t, J=1.2Hz, 1H), 7.01 (t, J=8.68 Hz, 1H), 7.16 (d, J=8.48 Hz, 2H), 7.26 (d,J=8.96 Hz, 1H), 7.65-7.72 (m, 3H), 8.08 (d, J=3.72 Hz, 1H), 9.20 (s,1H), 9.34 (s, 1H); LCMS: m/e 469.3 (M+1).

Example 212 Preparation of1-(3-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-2-methylprop-2-en-1-oneI-322

The title compound was prepared according to the schemes, steps andintermediates described in Example 112, by using1,3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2 andisopropenylmagnesium bromide in place of 8 in step 5. ¹H NMR (DMSO-d₆) δppm: 1.96 (s, 3H), 3.30 (s, 3H), 3.63 (t, J=4.6 Hz, 2H), 4.07 (t, J=4.36Hz, 2H), 5.62 (s, 1H), 6.00 (s, 1H), 6.99 (t, J=9.24 Hz, 1H), 7.26 (d,J=8.92 Hz, 1H), 7.39 (d, J=7.56 Hz, 1H), 7.46 (t, J=7.72 Hz, 1H), 7.62(bd, J=14.4 Hz, 1H), 7.93 (s, 1H), 8.12-8.14 (m, 2H), 9.25 (s, 1H), 9.58(s, 1H); LCMS: m/e 441.2 (M+1).

Example 213 Preparation of1-(3-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-3-methylbut-3-en-2-oneI-328

The title compound was prepared according to the schemes, steps andintermediates described in Example 112, by using ethyl3-aminophenylacetate in place of 2 in step 1,3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2 andisopropenylmagnesium bromide in place of 8 in step 5. ¹H NMR (DMSO-d₆) δppm: 1.8 (s, 3H), 3.31 (s, 3H), 3.64 (t, J=4.56 Hz, 2H), 4.06 (s, 2H),4.09 (t, J=4.37 Hz, 2H), 5.95 (s, 1H), 6.23 (s, 1H), 6.92 (d, J=7.52 Hz,1H), 7.02 (t, J=9.4 Hz, 1H), 7.27 (t, J=7.8 Hz, 2H), 7.50 (s, 1H),7.66-7.72 (m, 2H), 8.10 (d, J=3.56 Hz, 1H), 9.21 (s, 1H), 9.36 (s, 1H);LCMS: m/e 455.1 (M+1).

Example 214 Preparation of2-((3-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)(hydroxy)methyl)acrylonitrileI-326

The title compound was prepared according to the schemes, steps andintermediates described in Example 107, by using3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2. ¹H NMR(DMSO-d₆) δ ppm: 3.30 (s, 3H), 3.62-3.65 (m, 2H), 4.09 (t, J=4.6 Hz,2H), 5.29 (d, J=3.84 Hz, 1H), 6.13 (s, 1H), 6.19 (s, 1H), 6.31 (d,J=4.04 Hz, 1H), 7.03 (t, J=9.24 Hz, 1H), 7.10 (d, J=7.44 Hz, 1H), 7.28(d, J=8.72 Hz, 1H), 7.36 (t, J=7.8 Hz, 1H), 7.62 (s, 1H), 7.66 (dd,J=2.32 & 14.44 Hz, 1H), 7.89 (d, J=8.2 Hz, 1H), 8.10 (d, J=3.68 Hz, 1H),9.14 (s, 1H), 9.45 (s, 1H); LCMS: m/e 454 (M+1).

Example 215 Preparation of2-((4-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)(hydroxy)methyl)acrylonitrileI-327

The title compound was prepared according to the schemes, steps andintermediates described in Example 107, by using methyl 4-aminobenzoatein place of 2 in step 1 and 3-fluoro-4-(2-methoxyethoxy)aniline in placeof 4 in step 2. ¹H NMR (DMSO-d₆) δ ppm: 3.30 (s, 3H), 3.64 (dd, J=2.96 &4.56 Hz, 2H), 4.08 (t, J=4.48 Hz, 2H), 5.29 (d, J=3.8 Hz, 1H), 6.11 (s,1H), 6.21 (s, 1H), 6.24 (d, J=4.12 Hz, 1H), 7.01 (t, J=9.4 Hz, 1H),7.29-7.34 (m, 3H), 7.68 (dd, J=2.2 & 14.16 Hz, 1H), 7.77 (d, J=8.52 Hz,2H), 8.11 (d, J=3.68 Hz, 1H), 9.23 (s, 1H), 9.42 (s, 1H); LCMS: m/e454.0 (M+1).

Example 216 Preparation ofN-(3-(2-(4-chloro-3-(2-hydroxy-2-methylpropoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-249

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using4-chloro-3-(2-hydroxy-2-methylpropoxy)aniline in place of 4 in step 2.¹H NMR (DMSO-d₆) δ ppm: 1.20 (s, 6H), 3.61 (s, 2H), 4.61 (s, 1H), 5.75(d, J=11.4 Hz, 1H), 6.24 (d, J=18.36 Hz, 1H), 6.44 (dd, J=10.32 & 17.08Hz, 1H), 7.13 (d, J=8.64 Hz, 1H), 7.28 (t, J=8 Hz, 1H), 7.37-7.44 (m,3H), 7.55 (d, J=7.08 Hz, 1H), 7.93 (s, 1H), 8.12 (d, J=3.44 Hz, 1H),9.23 (s, 1H), 9.47 (s, 1H), 10.11 (s, 1H); LCMS: m/e 472.0 (M+1).

Example 217 Preparation ofN-(3-(5-fluoro-2-(3-fluoro-4-(2-hydroxy-2-methylpropoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-315

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-fluoro-4-(2-hydroxy-2-methylpropoxy)aniline in place of 4 in step 2.¹H NMR (DMSO-d₆) δ ppm: 1.19 (s, 6H), 3.67 (s, 2H), 4.62 (s, 1H), 5.75(d, J=10.4 Hz, 1H), 6.25 (d, J=17.2 Hz, 1H), 6.45 (dd, J=10 & 16.8 Hz,1H), 6.94 (t, J=9.2 Hz, 1H), 7.29 (t, J=8 Hz, 2H), 7.43 (d, J=8.4 Hz,1H), 7.49 (d, J=7.6 Hz, 1H), 7.67 (d, J=13.6 Hz, 1H), 7.94 (s, 1H), 8.11(d, J=3.6 Hz, 1H), 9.19 (s, 1H), 9.45 (s, 1H), 10.14 (s, 1H); LCMS: m/e456 (M−1).

Example 218 Preparation ofN-(3-(5-fluoro-2-(3-fluoro-4-(1-hydroxypropan-2-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-333

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-fluoro-4-(2-hydroxy-1-methylethoxy)aniline in place of 4 in step 2. ¹HNMR (DMSO-d₆) δ ppm: 1.16 (d, J=6.12 Hz, 3H), 3.40-3.46 (m, 1H),3.50-3.56 (m, 1H), 4.22 (sextet, J=5.6 Hz, 1H), 4.84 (t, J=5.68 Hz, 1H),5.75 (dd, J=1.96 & 10.08 Hz, 1H), 6.25 (dd, J=1.92 & 16.92 Hz, 1H), 6.46(dd, J=10.08 & 16.92 Hz, 1H), 6.98 (t, J=9.32 Hz, 1H), 7.26-7.31 (m,2H), 7.43 (d, J=8.76 Hz, 1H), 7.49 (d, J=8 Hz, 1H), 7.68 (dd, J=2.44 &14.28 Hz, 1H), 7.95 (s, 1H), 8.11 (d, J=3.68 Hz, 1H), 9.23 (s, 1H), 9.46(s, 1H), 10.17 (s, 1H); LCMS: m/e 442.2 (M+1).

Example 219 Preparation ofN-(3-(2-(4-(2,3-dihydroxypropoxy)-3-fluorophenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-334

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using4-(2,3-dihydroxypropoxy)-3-fluoroaniline in place of 4 in step 2. ¹H NMR(DMSO-d₆) δ ppm: 3.42 (t, J=5.6 Hz, 2H), 3.7-3.8 (m, 1H), 3.8-3.9 (m,1H), 3.94 (dd, J=4.36 & 9.92 Hz, 1H), 4.65 (t, J=5.64 Hz, 1H), 4.93 (d,J=5.08 Hz, 1H), 5.7-5.8 (m, 1H), 6.24 (dd, J=1.64 & 16.84 Hz, 1H), 6.44(dd, J=10 & 16.96 Hz, 1H), 6.94 (t, J=9.32 Hz, 1H), 7.28 (t, J=7.96 Hz,2H), 7.40 (d, J=8.28 Hz, 1H), 7.49 (d, J=7.44 Hz, 1H), 7.66 (d, J=14.24Hz, 1H), 7.92 (s, 1H), 8.09 (d, J=3.6 Hz, 1H), 9.17 (s, 1H), 9.45 (s,1H), 10.15 (s, 1H); LCMS: m/e 456 (M−1).

Example 220 Preparation ofN-(3-(2-(4-chloro-3-(1-hydroxy-2-methylpropan-2-yloxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-336

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using4-chloro-3-(1-hydroxy-2-methylpropan-2-yloxy)aniline in place of 4 instep 2. ¹H NMR (DMSO-d₆) δ ppm: 1.22 (s, 6H), 3.47 (d, J=5.88 Hz, 2H),4.88 (t, J=5.84 Hz, 1H), 5.75 (dd, J=3.24 & 10 Hz, 1H), 6.25 (dd, J=2 &16.92 Hz, 1H), 6.46 (dd, J=10.12 & 17.08 Hz, 1H), 7.15 (d, J=8.8 Hz,1H), 7.31 (t, J=8.2 Hz, 1H), 7.40-7.45 (m, 1H), 7.51-7.60 (m, 3H), 7.93(s, 1H), 8.13 (d, J=3.56 Hz, 1H), 9.24 (s, 1H), 9.47 (s, 1H), 10.12 (s,1H); LCMS: m/e 472.2 (M+1).

Example 221 Preparation ofN-(3-(2-(4-chloro-3-(1-hydroxypropan-2-yloxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-337

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using4-chloro-3-(1-hydroxypropan-2-yloxy)aniline in place of 4 in step 2. ¹HNMR (DMSO-d₆) δ ppm: 1.18 (d, J=6.12 Hz, 3H), 3.40-3.47 (m, 1H),3.50-3.56 (m, 1H), 4.20-4.30 (m, 1H), 4.82 (t, J=5.6 Hz, 1H), 5.75 (dd,J=1.88 & 10.08 Hz, 1H), 6.25 (dd, J=1.92 & 16.92 Hz, 1H), 6.45 (dd,J=10.08 & 16.92 Hz, 1H), 7.12 (d, J=8.76 Hz, 1H), 7.29 (t, J=8.08 Hz,1H), 7.40-7.44 (m, 3H), 7.52 (d, J=8.44 Hz, 1H), 7.91 (s, 1H), 8.12 (d,J=3.64 Hz, 1H), 9.21 (s, 1H), 9.45 (s, 1H), 10.12 (s, 1H); LCMS: m/e458.0 (M+1).

Example 222 Preparation ofN-(3-(2-(4-(2,3-dihydroxypropoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-335

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using4-(1-hydroxypropan-2-yloxy)aniline in place of 4 in step 2. ¹H NMR(DMSO-d₆) δ ppm: 3.43 (dd, J=0.64 & 6.84 Hz, 2H), 3.70-3.80 (m, 2H),3.85-3.95 (m, 1H), 4.62 (t, J=5.6 Hz, 1H), 4.88 (d, J=4.76 Hz, 1H), 5.75(dd, J=1.76 & 10.08 Hz, 1H), 6.25 (dd, J=1.72 & 16.92 Hz, 1H), 6.45 (dd,J=10.08 & 16.88 Hz, 1H), 6.74 (d, J=9 Hz, 2H), 7.27 (t, J=8.08 Hz, 1H),7.39 (d, J=8.04 Hz, 1H), 7.38-7.53 (m, 3H), 7.93 (s, 1H), 8.05 (d,J=3.68 Hz, 1H), 8.93 (s, 1H), 9.35 (s, 1H), 10.11 (s, 1H); LCMS: m/e440.3 (M+1).

Example 223 Preparation of(R)—N-(3-(2-(4-chloro-3-(tetrahydrofuran-3-yloxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-341

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(R)-4-chloro-3-(tetrahydrofuran-3-yloxy)aniline in place of 4 in step 2.¹H NMR (DMSO-d₆) δ ppm: 1.85-1.95 (m, 1H), 2.0-2.15 (m, 1H), 3.60-3.70(m, 1H), 3.73-3.83 (m, 3H), 4.68 (s, 1H), 5.74 (dt, J=1.92 & 10.0 Hz,1H), 6.23 (dd, J=1.88 & 16.92 Hz, 1H), 6.43 (dd, J=10.12 & 16.96 Hz,1H), 7.14 (d, J=8.72 Hz, 1H), 7.29 (t, J=8.08 Hz, 1H), 7.33 (dd, J=4.16& 8.76 Hz, 1H), 7.41-7.47 (m, 3H), 7.90 (s, 1H), 8.13 (d, J=3.56 Hz,1H), 9.28 (s, 1H), 9.47 (s, 1H), 10.13 (s, 1H); LCMS: m/e 469.8 (M+1).

Example 224 Preparation ofN-(3-(5-fluoro-2-(3-fluoro-4-(1-hydroxy-2-methylpropan-2-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-332

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-fluoro-4-(1-hydroxy-2-methylpropan-2-yloxy)aniline in place of 4 instep 2. ¹H NMR (DMSO-d₆) δ ppm: 1.13 (s, 6H), 3.36 (d, J=5.84 Hz, 2H),4.86 (t, J=5.84 Hz, 1H), 5.73 (dd, J=1.96 & 10.04 Hz, 1H), 6.24 (dd,J=1.96 & 16.96 Hz, 1H), 6.44 (dd, J=10.08 & 16.92 Hz, 1H), 6.95 (t,J=9.16 Hz, 1H), 7.23 (dd, J=1.64 & 8.96 Hz, 1H), 7.28 (t, J=8.12 Hz,1H), 7.43 (d, J=8.8 Hz, 1H), 7.48 (d, J=7.92 Hz, 1H), 7.70 (dd, J=2.48 &13.84 Hz, 1H), 7.92 (s, 1H), 8.11 (d, J=3.68 Hz, 1H), 9.25 (s, 1H), 9.45(s, 1H), 10.10 (s, 1H); LCMS: m/e 456.2 (M+1).

Example 225 Preparation ofN-(3-(2-(3-(2,3-dihydroxypropoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-339

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-(2,3-dihydroxypropoxy)aniline in place of 4 in step 2. ¹H NMR (MeOD) δppm: 3.59-3.69 (m, 2H), 3.88-3.98 (m, 3H), 5.78 (dd, J=2.16 & 9.6 Hz,1H), 6.36 (dd, J=2.24 & 17.04 Hz, 1H), 6.44 (dd, J=9.56 & 16.96 Hz, 1H),6.54-6.57 (m, 1H), 7.08-7.12 (m, 2H), 7.32 (t, J=7.92 Hz, 2H), 7.44 (dd,J=7.88 & 13.4 Hz, 2H), 7.94 (d, J=3.8 Hz, 1H), 8.09 (s, 1H); LCMS: m/e440.1 (M+1).

Example 226 Preparation ofN-(4-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-351

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by usingtert-butoxycarbonylamino-4-aminoaniline in place of 2 in step 1 and3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2. ¹H NMR(DMSO-d₆) δ ppm: 3.30 (s, 3H), 3.63 (t, J=4.6 Hz, 2H), 4.08 (t, J=4.48Hz, 2H), 5.74 (dd, J=2 & 10.08 Hz, 1H), 6.25 (dd, J=1.96 & 16.92 Hz,1H), 6.44 (dd, J=10.04 & 16.96 Hz, 1H), 7.02 (t, J=9.48 Hz, 1H), 7.23(bd, J=7.44 Hz, 1H), 7.64 (d, J=9 Hz, 2H), 7.70-7.74 (m, 3H), 8.07 (d,J=3.72 Hz, 1H), 9.19 (s, 1H), 9.34 (s, 1H), 10.13 (s, 1H); LCMS: m/e442.0 (M+1).

Example 227 Preparation of2-((3-(5-fluoro-2-(6-(2-hydroxy-2-methylpropoxy)pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)(hydroxy)methyl)acrylonitrileI-312

The title compound was prepared according to the schemes, steps andintermediates described in Example 107, by using3-amino-6-(2-hydroxy-2-methylpropoxy)pyridine in place of 4 in step 2.¹H NMR (DMSO-d₆) δ ppm: 1.18 (s, 6H), 3.98 (s, 2H), 4.61 (s, 1H), 5.31(d, J=3.88 Hz, 1H), 6.13 (s, 1H), 6.19 (s, 1H), 6.32 (d, J=4 Hz, 1H),6.75 (d, J=8.88 Hz, 1H), 7.10 (d, J=7.72 Hz, 1H), 7.33 (t, J=7.84 Hz,1H), 7.68 (s, 1H), 7.84 (d, J=7.52 Hz, 1H), 7.96 (dd, J=2.72 & 8.88 Hz,1H), 8.08 (d, J=3.64 Hz, 1H), 8.33 (d, J=1.76 Hz, 1H), 9.06 (s, 1H),9.44 (s, 1H); LCMS: m/e 451 (M+1).

Example 228 Preparation of4-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)phenoxy)-N-methylpicolinamideI-342

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using4-(4-aminophenoxy)-N-methylpicolinamide in place of 4 in step 2. LC/MS(M+H) 500.2

Example 229 Preparation of(R)-1-(3-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-344

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(R)-1-tert-butoxycarbonyl-3-aminopiperidine in place of 2 in step 1 and3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2. LC/MS (M+H)434.1.

Example 230 Preparation of(R)-1-(3-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-345

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(R)-1-tert-butoxycarbonyl-3-aminopiperidine in place of 2 in step 1 and4-(2-methoxyethoxy)aniline in place of 4 in step 2. LC/MS (M+H) 416.2

Example 231 Preparation of4-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)phenoxy)pyridine I-346

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using4-(4-aminophenoxy)pyridine in place of 4 in step 2. LC/MS(RT=2.802/(M+H)) 500.2

Example 232 Preparation of1-((R)-3-(5-fluoro-2-(4-((S)-tetrahydrofuran-3-yloxy)phenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-347

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(R)-1-tert-butoxycarbonyl-3-aminopiperidine in place of 2 in step 1 and4-(S)-(tetrahydrofuran-3-yloxy)aniline in place of 4 in step 2. LC/MS(M+H) 428.3.

Example 233 Preparation of1-((R)-3-(5-fluoro-2-(4-((R)-tetrahydrofuran-3-yloxy)phenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-oneI-348

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(R)-1-tert-butoxycarbonyl-3-aminopiperidine in place of 2 in step 1 and4-(R)-(tetrahydrofuran-3-yloxy)aniline in place of 4 in step 2. LC/MS(M+H) 428.3.

Example 234 Preparation ofN-(3-(2-(2,3-dihydrobenzo[b]1,4]dioxin-6-ylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-349

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using6-amino-2,3-dihydrobenzo[b]1,4]dioxane in place of 4 in step 2. LC/MS(M+H) 408.

Example 235 Preparation of1-(6-(4-(3-chloro-4-(pyridine-2-ylmethoxy)phenylamino)-5-fluoropyrimidin-2-ylamino)-2H-benzo[b][1,4]oxazin-4(3H)-yl)prop-2-en-1-oneI-343

The title compound was prepared according to the schemes, steps andintermediates described in Example 35, using3-chloro-4-(pyridine-2-ylmethoxy)aniline in place of 2 in step 1. LC/MS(M+H) 533.1.

Example 236 Preparation ofN-(3-(5-cyano-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-350

The title compound was prepared according to the schemes, steps andintermediates described in Example 94, by using3-fluoro-4-(2-methoxyethoxy)aniline for 4 in step 2. LC/MS (M+H) 449.1

Example 237 Preparation ofN-(3-(5-trifluoromethyl-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-352

The title compound was prepared according to the schemes, steps andintermediates described in Example 189 using3-fluoro-4-(2-methoxyethoxy)aniline for 2 in step 1. LC/MS (M+H) 492.1

Example 238 Preparation ofN-(3-(2-(4-chloro-3-(2-methoxyethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-321

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using4-chloro-3-(2-methoxyethoxy)aniline in place of 4 in step 2. ¹H NMR(DMSO-d₆) δ ppm: 3.30 (s, 3H), 3.60 (t, J=4.56 Hz, 2H), 3.88 (t, J=3.48Hz, 2H), 5.74 (dd, J=4.36 & 10.0 Hz, 1H), 6.24 (dd, J=1.8 & 16.88 Hz,1H), 6.44 (dd, J=4.36 & 10.0 Hz, 1H), 7.13 (d, J=8.72 Hz, 1H), 7.28 (t,J=8.04 Hz, 1H), 7.33 (dd, J=2.16 & 8.8 Hz, 1H), 7.41 (d, J=7.96 Hz, 1H),7.47-7.49 (m, 2H), 7.84 (s, 1H), 8.13 (d, J=3.6 Hz, 1H), 9.27 (s, 1H),9.47 (s, 1H), 10.12 (s, 1H); LCMS: m/e 458.0 (M+1).

Example 239 Preparation ofN-(3-(5-fluoro-2-(6-(2-hydroxy-2-methylpropoxy)pyridin-3-ylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-313

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-amino-6-(2-hydroxy-2-methylpropoxy)pyridine in place of 4 in step 2.¹H NMR (DMSO-d₆) δ ppm: 1.16 (s, 6H), 3.93 (s, 2H), 4.57 (s, 1H), 5.74(dd, J=1.68 & 10.04 Hz, 1H), 6.24 (dd, J=1.84 & 16.92 Hz, 1H), 6.45 (dd,J=10.04 & 16.88 Hz, 1H), 6.65 (d, J=8.88 Hz, 1H), 7.26 (t, J=8.04 Hz,1H), 7.39 (d, J=8 Hz, 1H), 7.48 (d, J=7.8 Hz, 1H), 7.91 (s, 1H), 7.99(dd, J=2.72 & 8.92 Hz, 1H), 8.07 (d, J=3.68 Hz, 1H), 8.27 (d, J=2.52 Hz,1H), 9.06 (s, 1H), 9.41 (s, 1H), 10.1 (s, 1H); LCMS: m/e 439.0 (M+1).

Example 240 Preparation of N-(3-(5-fluoro-2-(3-fluoro-4-(3-(methylsulfonyl)propoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide I-318

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using3-fluoro-4-(3-(methylsulfonyl)propoxy)aniline in place of 4 in step 2.¹H NMR (DMSO-d₆) δ ppm: 2.05-2.15 (m, 2H), 3.01 (s, 3H), 3.24 (t, J=7.56Hz, 2H), 4.05 (t, J=6.12 Hz, 2H), 5.74 (dd, J=1.84 & 9.72 Hz, 1H), 6.24(dd, J=1.72 & 16.96 Hz, 1H), 6.44 (dd, J=10 & 16.84 Hz, 1H), 6.96 (t,J=9.36 Hz, 1H), 7.28 (t, J=8.04 Hz, 2H), 7.40 (d, J=7.8 Hz, 1H), 7.48(d, J=8.32 Hz, 1H), 7.69 (dd, J=2.2 & 14.4 Hz, 1H), 7.91 (s, 1H), 8.10(d, J=3.64 Hz, 1H), 9.20 (s, 1H), 9.44 (s, 1H), 10.12 (s, 1H); LCMS: m/e504.2 (M+1).

Example 241 Preparation of1-(3-(5-fluoro-2-(3-fluoro-4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-4-methylpent-3-en-2-oneI-330

The title compound was prepared according to the schemes, steps andintermediates described in Example 112, by using ethyl4-aminomethylbenzoate in place of 2 in step 1 and3-fluoro-4-(2-methoxyethoxy)aniline in place of 4 in step 2. ¹H NMR(DMSO-d₆) δ ppm: 1.83 (s, 3H), 2.05 (s, 3H), 3.31 (s, 3H), 3.64 (t,J=4.56 Hz, 2H), 3.69 (s, 2H), 4.08 (t, J=4.4 Hz, 2H), 6.18 (s, 1H), 6.92(d, J=7.44 Hz, 1H), 7.01 (t, J=9.36 Hz, 1H), 7.27 (t, J=7.84 Hz, 2H),7.51 (s, 1H), 7.64-7.71 (m, 2H), 8.09 (d, J=3.64 Hz, 1H), 9.19 (s, 1H),9.34 (s, 1H); LCMS: m/e 469.1 (M+1).

Example 242 Preparation ofN-(3-(2-(4-chloro-3-(2,3-dihydroxypropoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-353

The title compound was prepared according to the schemes, steps andintermediates described below.

A) Pd(OAc)₂, BINAP, Cs₂CO₃, toluene, 110° C. 16 h; B) TFA, CH₂Cl₂, rt, 2h; C) acryloyl chloride, K₂CO₃, NMP, rt, 45 min.

Step-1

A solution of 2 (200 mg, 0.77 mmol), 1 (262 mg, 0.77 mmol), Pd(OAc)₂(17.3 mg, 0.07 mmol), BINAP (24 mg, 0.038 mmol) and Cs₂CO₃ (630 mg, 1.9mmol) in degassed toluene (toluene was purged with N₂ for 30 min) washeated for 16 h at 100° C. under N₂ atmosphere. The reaction mixture wascooled, diluted with EtOAc (15 mL) and filtered through Celite®. Thefiltrate was washed with water (5 mL) and brine (3 mL), dried overNa₂SO₄, filtered, and concentrated under reduced pressure to give 3 (0.3g, 69%) as a yellow solid.

Step-2

To a stirred solution of 3 (300 mg, 0.5 mmol) in dry CH₂Cl₂ (6 mL) at 0°C. was added CF₃COOH (3 mL), and the reaction mixture was kept at thistemperature for 30 min. The reaction was allowed to come to rt andstirred at this temperature for 3 h. The reaction mixture wasconcentrated under reduced pressure, and the residue was quenched withwater (5 mL), basified with NaCO₃ solution, and extracted with ethylacetate (2×10 mL). The combined extracts were washed with water (5 mL)and brine (5 mL), dried over Na₂SO₄, and concentrated under reducedpressure to get 4 (200 mg, 88%) as a yellow solid.

Step-3

To a stirred solution of 4 (240 mg, 0.5 mmol), in NMP (1.5 mL) at 0° C.was added potassium carbonate (780 mg, 5.7 mmol) and acryloyl chloride(57 mg, 0.5 mmol), and the reaction mixture was stirred at 0° C. for 3h. The reaction mixture was further stirred at rt for 30 min andquenched by dropwise addition to a cold, stirring solution of 10% NaHCO₃and stirred at 0° C. for 30 min. A solid precipitated out and wasisolated by filtration through a Buchner funnel. The solid was washedwith cold water, dissolved in EtOAc (20 mL) and was basified by usingtriethylamine and washed with water (2 mL), brine (1 mL), dried overNa₂SO₄ and concentrated under reduced pressure. The residue was purifiedby preparative HPLC to give the title compound (45 mg, 15.5%) as a whitesolid. ¹H NMR (DMSO-d6) δ ppm: 3.43-3.50 (m, 2H), 3.78-3.85 (m, 2H),3.89-3.92 (m, 1H), 4.65 (t, J=5.6 Hz, 1H), 4.93 (d, J=4.8 Hz, 1H), 5.76(dd, J=1.92 & 10.04 Hz, 1H), 6.26 (dd, J=1.92 & 16.92 Hz, 1H), 6.46 (dd,J=10.08 & 16.92 Hz, 1H), 7.14 (d, J=8.72 Hz, 1H), 7.30 (t, J=8.08 Hz,1H), 7.39-7.43 (m, 2H), 7.46 (dd, J=2.2 & 8.72 Hz, 1H), 7.56 (d, J=8.04Hz, 1H), 7.94 (s, 1H), 8.14 (d, J=3.6 Hz, 1H), 9.24 (s, 1H), 9.48 (s,1H), 10.13 (s, 1H); LCMS: m/e 473.8 (M+).

Synthesis of Intermediate 2

A) DIAD, PPh₃, Et₃N, dry THF, rt, 1 h; B) H₂, Ra Ni, methanol, 2 h.

Step-1

To a stirred solution of 2′ (0.640 g, 3.7 mmol) in THF (20 mL) wereadded 1′ (0.5 g, 3.7 mmol), PPh₃ (1.09 g, 4.1 mmol) and Et₃N (0.73 g,5.6 mmol) under N₂ atmosphere. The reaction mixture was cooled to 0° C.and DIAD (0.84 g, 4.1 mmol) was added. The reaction mixture was allowedto come to rt and stir for 1 h. The reaction was quenched with water,extracted with ethyl acetate (3×10 mL), and the combined extracts werewashed with water and brine solution (5 mL each). The residue obtainedafter concentration under reduced pressure was purified by columnchromatography (SiO₂, 60-120, pet ether/ethyl acetate, 9/1) to give 3′(0.6 g, 60%) as a white solid.

Step-2

To a solution of 3′ (0.3 g, 1.04 mmol) in methanol was added Raney Ni(60 mg, 20% w/w) under N₂, and the reaction mixture was kept under H₂atmosphere (bladder pressure) for 16 h. The reaction mixture wasfiltered through a bed of Celite®, and the filtrate was concentratedunder reduced pressure. The residue was diluted with 1.5 N HCl (2 mL)and washed with ethyl acetate (5 mL) to remove organic impurities. Theaqueous layer was basified with NaHCO₃ solution (5 mL), extracted withethyl acetate, washed with water (2 mL) and brine (2 mL), and dried overanhydrous Na₂SO₄. Filtration followed by concentration under reducedpressure gave 2 (0.2 g, 76.9%) as a brown liquid.

Example 243 Preparation of(S)—N-(3-(2-(4-chloro-3-(tetrahydrofuran-3-yloxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-354

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(S)-4-chloro-3-(tetrahydrofuran-3-yloxy)aniline in place of 4 in step 2.LCMS: m/e 469.8 (M+1).

Example 244 Preparation of(N-(3-(5-fluoro-2-(3-fluoro-4-(((2S,4R)-4-hydroxypyrrolidin-2-yl)methoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-355

The title compound was prepared according to the schemes, steps andintermediates described below.

A) Pd(OAc)₂, BINAP, Cs₂CO₃, toluene, 110° C., 6 h; B) TFA, CH₂Cl₂, rt, 1h; C) (Boc)₂O, 30 min then acryloyl chloride, K₂CO₃, NMP, 0° C., 90 min;D) HF (49% aq. Solution), CH₃CN, rt, 2 h; E) TFA, DCM, rt, 2 h.

Step-1

A solution of 2 (0.50 g, 1.13 mmol), 1 (0.30 g, 1.13 mmol), Pd(OAc)₂(0.0025 g, 0.1 mmol), BINAP (0.0035 g, 0.05 mmol) and Cs₂CO₃ (0.92 g,2.8 mmol) in degassed toluene (toluene was purged with N₂ for 30 min)was heated at 110° C. for 16 h under N₂ atmosphere. The reaction mixturewas cooled, diluted with EtOAc (20 mL), washed with water (10 mL), brine(10 mL) and dried over Na₂SO₄. Filtration followed by concentrationunder reduced pressure offered a residue which was further washed withhexane to give 3 (0.3 g, 42.8%) as a yellow solid.

Step-2

To a solution of 3 (0.3 g, 0.44 mmol) in methanol (5 mL)) was added Pd/C(0.030 g, 10% w/w) and the reaction mixture was allowed to stir under H₂atmosphere (balloon) at rt for 16 h. The reaction mixture was filteredthrough a pad of Celite® and was concentrated under reduced pressure togive 4 (0.19 g, 67.6%) as a yellow solid.

Step-3

To a stirred solution of 4 (0.1 g, 0.15 mmol) in NMP (1.0 mL) at rt wasadded Boc anhydride (0.046 g, 0.212 mmol) and the reaction mixture wasstirred at rt for 60 min. It was then cooled to 0° C. and to it wasadded K₂CO₃ (0.107 g, 0.77 mmol), acryloyl chloride (0.016 g, 0.18 mmol)and the reaction mixture was stirred at 0° C. for 90 min. The reactionmixture was added drop wise, to a cold, stirring solution of 10% NaHCO₃.After the addition was over, the solution was stirred for another 30 minat 0° C., and the solid was isolated by filtration through a Buchnerfunnel. The solid was washed with cold water, hexane and was dissolvedin methanol:dichloromethane (50:50, 10 mL) and concentrated underreduced pressure. The residue obtained was suspended in cold water (5mL), Et₃N was added to it and it was extracted with ethyl acetate (2×10mL). The combined ethyl acetate extract was washed with water (5 mL),brine (5 mL), dried over Na₂SO₄ and concentrated under reduced pressure.The residue was further purified by column chromatography (SiO₂,methanol/chloroform: 4/96) to give 5 (0.075 g, 71.4%) as yellow solid.

Step-4

To a solution of 5 (15 mg, 0.02 mmol) in acetonitrile was added HF (49%aq. Solution, 0.0048 mL, 0.024 mmol) at 0° C. The reaction mixturestirred at rt for 2 h, was extracted with ethyl acetate (2 mL), waswashed with water (1 mL), and was dried over Na₂SO₄ and filtered. Thefiltrate was concentrated under reduced pressure. The residue showed 60%purity by LCMS and was used in the next step without furtherpurification.

Step-5

To a stirred solution of 6 (0.008 g, 0.013 mmol) in CH₂Cl₂ (0.024 mL)was added TFA (0.016 mL) at 0° C. The reaction mixture was allowed tocome to rt and was stirred for additional 2 h. It was then concentratedand was stirred with cold 10% NaHCO₃ (1.0 mL). It was extracted withEtOAc (2×2 mL) and the combined EtOAc extract was washed with brine (1mL), was dried over Na₂SO₄ and was concentrated under reduced pressure.The crude residue was further purified by column chromatography (SiO₂,methanol/chloroform: 2/98) and was then purified by preparative TLC togive the title compound (2 mg, 81% purity by HPLC, and 79% purity byLCMS) as a white solid. LCMS: m/e 483 (M+).

Compound 2 was prepared according to the schemes, steps andintermediates described below.

A) MeOH, SOCl₂, reflux, 5 h; B) (Boc)₂O, Et₃N, CH₂Cl₂, rt, 5 h; C)TBDMS-Cl, imidazole, DMF, rt, 16 h; D) LAH solution (1M in THF), −20°C., 20 min; E) DIAD, PPh₃, Et₃N, THF, 16 h; F) H₂, Pd/C, methanol, rt,16 h.

Step-1

To a stirred solution of 1′ (2 g, 15.26 mmol) was added a solutionprepared by adding thionyl chloride (2 mL) to methanol (20 mL). Thereaction mixture was heated at reflux for 5 h. After completion of thereaction, methanol was removed under reduced pressure to give 2′ ascolorless salt (3.0 g) it was used as such in the next reaction.

Step-2

To a stirred solution of 2′ (3.0 g, 12.24 mmol) in DCM (30 mL) was addedEt₃N (1.85 g, 18.31 mmol) and Boc anhydride (2.92 g, 13.46 mmol).Stirring was continued at rt for 5 h after which the reaction wasquenched with water. The organic layer was separated, dried andconcentrated under reduced pressure. The residue was purified by columnchromatography (SiO₂, 60-120, 100% ethyl acetate) to give 3′ (3.2 g,64%) as a white solid.

Step-3

To a stirred solution of 3′ (3 g, 12.24 mmol) in DMF (30 mL) was addedimidazole (1.2 g, 18.36 mmol) followed by TBDMS chloride (1.84 g, 12.24g). Stirring was continued for 16 h. The reaction mixture was dilutedwith ethyl acetate (50 mL) and the ethyl acetate layer was separated. Itwas washed with water (5 mL), brine solution (5 mL) and dried overNa₂SO₄. Filtration followed by concentration under reduced pressureoffered a residue which was purified by column chromatography (SiO₂,60-120, petroleum ether/ethyl acetate: 6/4) to give 4′ (3.2 g, 80%) as acolorless liquid.

Step-4

To a stirred solution of 4′ (0.5 g, 1.39 mmol) in THF (5 mL) was addedLAH (1.39 mL, 1M solution, 1.39 mmol) at −20° C. The reaction wascontinued at the same temperature for 15 min after which it was quenchedwith Na₂SO₄ solution. The reaction mass was filtered through Celite® andfiltrate was concentrated under reduced pressure. The residue wasdiluted with ethyl acetate (10 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced under reduced pressure to give5′ (0.3 g, 65%) as a colorless liquid.

Step-5

To a stirred solution of 5′ (0.1 g, 0.3 mmol) in THF (6 mL) were added6′ (0.047 g, 0.3 mmol), PPh₃ (0.16 g, 0.64 mmol) and Et₃N (0.048 g, 0.48mmol) under N₂ atmosphere. The reaction mixture was cooled to 0° C. andto it was added DIAD (0.094 g, 0.48 mmol). The reaction mixture wasallowed to come to rt and stirred at it for 1 h. It was quenched withwater, was extracted with ethyl acetate (2×5 mL) and the combined ethylacetate extract was washed with water and brine solution (5 mL each).The residue obtained after concentration under reduced pressure waspurified by column chromatography (SiO₂, 60-120, pet ether/ethylacetate, 9/1) to give 7′ (0.120 g, 85%) as a yellow solid

Step-6

To a solution of 7′ (0.1 g, 0.21 mmol) in methanol (5 mL)) was addedPd/C (0.010 g, 10% w/w) and the reaction mixture was allowed to stirunder H₂ atmosphere (bladder) at rt for 16 h. The reaction mixture wasfiltered through a pad of Celite® and concentrated under reducedpressure to give 2 (0.085 g, 91%) as a brownish viscous oil. It was usedin the next step without further purification.

Example 245 Preparation of tert-butyl2-(2-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)-2-fluorophenoxy)ethoxy)ethylcarbamateI-356

The title compound was prepared according to the schemes, steps andintermediates described below.

A) Pd(OAc)₂, BINAP, Cs₂CO₃, toluene, 110° C., 6 h; B) TFA, CH₂Cl₂, RT 1h; C) (Boc)₂O, 30 min then acryloyl chloride, K₂CO₃, NMP, 0° C., 90 min.

Step-1

A solution of 2 (0.050 g, 0.159 mmol), 1 (0.053 g, 0.159 mmol), Pd(OAc)₂(0.0035 g, 0.01590 mmol), BINAP (0.0049 g, 0.0079 mmol) and Cs₂CO₃(0.129 g, 0.3975 mmol) in degassed toluene (toluene was purged with N₂for 30 min) was heated at 110° C. for 16 h under N₂ atmosphere. Thereaction mixture was cooled, diluted with EtOAc (20 mL), washed withwater (10 mL), brine (10 mL) and dried over Na₂SO₄. Filtration followedby concentration under reduced pressure offered a residue which wasfurther washed with hexane to give 3 (0.049 g, 50%) as a brown solid.

Step-2

To a stirred solution of 3 (0.047 g, 0.0762 mmol) in dry CH₂Cl₂ (3 mL)at 0° C. was added CF₃COOH (1.0 mL) and the reaction mixture was stirredat 0° C. for 30 min. The reaction was allowed to come to rt and stirredat it for 1 h. It was concentrated under reduced pressure and theresidue was quenched with NaHCO₃ solution (3 mL). The contents wereextracted with ethyl acetate (3×10 mL) and the combined EtOAc extractwas washed with water (10 mL) followed by 10% citric acid solution (3×10mL). The combined citric acid extract was basified with 10% NaOHsolution and extracted with EtOAc (3×25 mL). The EtOAc extract waswashed with water (20 mL), brine (10 mL) and dried over Na₂SO₄. to get 4(0.028 g, 88%) as a light yellow solid.

Step-3

To a stirred solution of 4 (0.028 g, 0.06731 mmol) in NMP (1.0 mL) at rtwas added (Boc)₂O (0.016 g, 0.07404 mmol) and the reaction mixture wasstirred at rt for 30 min. It was cooled to 0° C. and to it was addedK₂CO₃ (0.051 g, 0.372 mmol) and acryloyl chloride (0.0067 g, 0.07404mmol) and the reaction mixture was stirred at 0° C. for 30 min. Thereaction mixture was added dropwise, to a cold, stirring solution of 10%NaHCO₃. After the addition was over, the solution was stirred foranother 30 min at 0° C., and the solid was isolated by filtrationthrough a Buchner funnel. The solid was washed with cold water, hexaneand was dissolved in methanol:dichloromethane (50:50, 5 mL) andconcentrated under reduced pressure. The residue obtained was suspendedin cold water (3 mL), Et₃N was added to it and it was extracted withethyl acetate (2×5 mL). The combined ethyl acetate extract was washedwith water (5 mL), brine (5 mL), dried over Na₂SO₄ and concentratedunder reduced pressure to give the title compound (0.016 g, 42%) as agrey solid. ¹H NMR (DMSO-d6) δ ppm: 1.37 (s, 9H), 3.09 (d, J=5.5 Hz,2H), 3.43 (d, J=5.84 Hz, 2H), 3.69 (s, 2H), 4.05 (s, 2H), 5.75 (d,J=11.12 Hz, 1H), 6.25 (d, J=16.76 Hz, 1H), 6.46 (dd, J=10.12 & 16.84 Hz,1H), 6.81 (s, 1H), 6.96 (t, J=9.12 Hz, 1H), 7.24-7.31 (m, 2H), 7.43 (d,J=7.96 Hz, 1H), 7.49 (d, J=7.56 Hz, 1H), 7.68 (d, J=14 Hz, 1H), 7.94 (s,1H), 8.11 (d, J=3.24 Hz, 1H), 9.21 (s, 1H), 9.46 (s, 1H), 10.15 (s, 1H);LCMS: m/e 571.1 (M+1).

The intermediate 2 was prepared according to the schemes, steps andintermediates described below.

A) (Boc)₂O, aq. NaOH, rt, 16 h; B) DIAD, PPh₃, Et₃N, dry THF, rt, 1 h;C) H₂, Pd/C, ethanol, rt, 16 h.

Step-1

To a solution of NaOH (0.76 g, 0.019 mmol) in water (9.6 mL) at rt wasadded 1′ (2.0 g, 19.022 mmol) and the reaction was stirred for 30 min. Asolution of Boc-anhydride (4.561 g, 20.92 mmol) in THF (12.0 mL) wasadded dropwise over 5 min to it. The reaction mixture was stirred at rtfor 16 h. It was concentrated under reduced pressure, diluted with water(20 mL) and extracted with EtOAc (4×50 mL). The combined EtOAc extractwas washed with water (50 mL), brine (50 mL), dried over Na₂SO₄ to give2′ (3.2 g, 82%) as a viscous oil.

Step-2

To a stirred solution of 2′ (0.38 g, 1.851 mmol) in THF (6 mL) wereadded 3′ (0.29 g, 1.851 mmol), PPh₃ (0.534 g, 2.0361 mmol) and Et₃N(0.280 g, 2.776 mmol) under N₂ atmosphere. The reaction mixture wascooled to 0° C. and to it was added DIAD (0.411 g, 2.0361 mmol). Thereaction mixture was allowed to come to rt and stirred at it for 1 h. Itwas quenched with water, extracted with ethyl acetate (3×5 mL) and thecombined ethyl acetate extract was washed with water and brine solution(5 mL each). The residue obtained after concentration under reducedpressure was purified by column chromatography (SiO₂, 60-120, petether/ethyl acetate, 9/1) to give 4′ (0.360 g, crude) as a yellow solid

Step-3

To a solution of 4′ (0.360 g, 1.0456 mmol) in ethanol (10 mL)) was addedPd/C (0.072 g, 20% w/w) and the reaction mixture was allowed to stirunder H₂ atmosphere (1.5 Kg hydrogen pressure) at rt for 16 h. Thereaction mixture was filtered through a pad of Celite® and concentratedunder reduced pressure to give 2 (0.28 g, 85%) as a brownish viscousoil. It was used in the next step without further purification.

Example 246 Preparation ofN¹-(2-(2-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)-2-fluorophenoxy)ethoxy)ethyl)-N⁵-(15-oxo-18-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azaoctadecyl)glutaramideI-362

The title compound was prepared according to the schemes, steps andintermediates described in Example 204, by using tert-butyl2-(2-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)-2-fluorophenoxy)ethoxy)ethylcarbamate(I-356, described in Example 245) in place of I-45 in step 1. ¹H NMR(DMSO-d₆) δ ppm: 10.1 (s, 1H), 9.87 (s, 1H), 9.52 (s, 1H), 8.09 (d,J=4.1 Hz, 1H), 7.86 (s, 1H), 7.78 (t, J=5.5 Hz, 1H), 7.66 (m, 3H), 7.48(dd, J=2.3 & 13.8 Hz, 1H), 7.33 (m, 2H), 7.21 (t, J=7.8 Hz, 1H), 7.11(d, J=9.2 Hz, 1H), 6.90 (t, J=9.2 Hz, 1H), 6.34 (m, 2H), 6.14 (dd, J=2.3& 17.0 Hz, 1H), 5.66 (dd, J=2.3 & 17.0 Hz, 1H), 4.20 (dd, J=5.0 & 7.3Hz, 1H), 3.99 (m, 3H), 3.61 (m, 2H), 3.12 (q, J=6.0 Hz, 2H), 2.97 (m,9H), 2.72 (m, 2H), 2.46 (m, 2H), 1.95 (m, 9H), 1.1-1.6 (m, 18H); LCMS:m/e 1013. (M+1).

Example 247 Preparation ofN-(3-(5-fluoro-2-(3-fluoro-4-(2-(2-methoxyethoxy)ethoxy)-phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-359

The title compound was prepared according to the schemes, steps andintermediates described below.

A) DIAD, PPh₃, Et₃N, dry THF, rt, 1 h; B) H₂, Pd/C, methanol, rt, 16 h;C) Pd(OAc)₂, BINAP, Cs₂CO₃, toluene, 110° C., 6 h; D) TFA, CH₂Cl₂, rt, 1h; E) (BOC)₂O, 30 min, then K₂CO₃, NMP, 0° C., 15 min.

Step-1

To a stirred solution of 1 (0.5 g, 3.18 mmol) in THF (10 mL) were added2 (0.38 g, 3.18 mmol), PPh₃ (0.91 g, 3.498 mmol) and Et₃N (0.48 g, 4.776mmol) under N₂ atmosphere. The reaction mixture was cooled to 0° C. andto it was added DIAD (0.707 g, 3.5 mmol). The reaction mixture wasallowed to come to rt and stirred at it for 1 h. It was quenched withwater, extracted with ethyl acetate (3×5 mL) and the combined EtOAcextract was washed with water and brine solution (5 mL each). Theresidue obtained after concentration under reduced pressure was purifiedby column chromatography (SiO₂, 60-120, pet ether/ethyl acetate, 7/3) togive 3 (0.61 g, 65%) as a white solid.

Step-2

To a solution of 3 (0.6 g, 2.31 mmol) in ethanol (20 mL)) was added Pd/C(0.060 g, 10% w/w) and the reaction mixture was allowed to stir under H₂atmosphere (bladder pressure) at rt for 16 h. The reaction mixture wasfiltered through a pad of Celite® and concentrated under reducedpressure to give 4 (0.375 g, 70.7%) as a brownish viscous oil.

Step-3

A solution of 4 (0.275 g, 1.19 mmol), 5 (0.403 g, 1.19 mmol), preparedaccording to Step-1 of Example 20, Pd(OAc)₂ (0.0026 g, 0.11 mmol), BINAP(0.0037 g, 0.059 mmol) and Cs₂CO₃ (0.969 g, 2.95 mmol) in degassedtoluene (toluene was purged with N₂ for 30 min) was heated at 110° C.for 16 h under N₂ atmosphere. The reaction mixture was cooled, dilutedwith EtOAc (20 mL), washed with water (10 mL), brine (10 mL) and driedover Na₂SO₄. Filtration followed by concentration under reduced pressureoffered a residue which was further purified by column chromatography(SiO₂, 60-120, pet ether/ethyl acetate 5/5) to give 6 (0.350 g, 55%) asa yellow solid.

Step-4

To a stirred solution of 6 (0.3 g, 0.56 mmol) in dry CH₂Cl₂ (3 mL) at 0°C. was added CF₃COOH (1.0 mL) and the reaction mixture was stirred at 0°C. for 30 min. The reaction was allowed to come to rt and stirred at itfor 1 h. It was concentrated under reduced pressure and the residue wasquenched with NaHCO₃ solution (3 mL) and extracted with EtOAc (3×25 mL).The combined EtOAc extract was washed with water (20 mL), brine (10 mL)and dried over Na₂SO₄ to give 7 (0.15 g, 62.5%) as a light brown viscousliquid.

Step-5

To a cooled solution of 7 (0.1 g, 0.23 mmol) in NMP (1.0 mL) at about 0°C. was added K₂CO₃ (0.15 g, 1.1 mmol), acryloyl chloride (0.0022 g, 0.25mmol) and the reaction mixture was stirred at 0° C. for 30 min. Thereaction mixture was added dropwise to a cold, stirring solution of 10%NaHCO₃. After the addition was over, the solution was stirred foranother 30 min at 0° C., and the solid was isolated by filtrationthrough a Buchner funnel. The solid was washed with cold water, hexaneand was dissolved in methanol:dichloromethane (50:50, 25 mL) andconcentrated under reduced pressure. The residue obtained was suspendedin cold water (3 mL), Et₃N was added to it and it was extracted withethyl acetate (2×5 mL). The combined ethyl acetate extract was washedwith water (5 mL), brine (5 mL), dried over Na₂SO₄ and concentratedunder reduced pressure to give the title compound (0.055 g, 50%) asyellow solid. ¹H NMR (DMSO-d6) δ ppm: 3.24 (s, 3H), 3.44 (t, J=4.88 Hz,2H), 3.57 (t, J=4.04 Hz, 2H), 3.69 (t, J=4.24 Hz, 2H), 4.04 (t, J=4.04Hz, 2H), 5.73 (d, J=10.12 Hz, 1H), 6.23 (d, J=16.8 Hz, 1H), 6.45 (dd,J=10.12 & 16.92 Hz, 1H), 6.95 (t, J=9.4 Hz, 1H), 7.28-7.30 (m, 2H), 7.42(d, J=8.04 Hz, 1H), 7.48 (d, J=7.48 Hz, 1H), 7.67 (d, J=14.36 Hz, 1H),7.93 (s, 1H), 8.10 (d, J=3.44 Hz, 1H), 9.20 (s, 1H), 9.44 (s, 1H), 10.14(s, 1H); LCMS: m/e 486.1 (M+1).

Example 248 Preparation of(S)—N-(3-(2-(4-chloro-3-(1-hydroxypropan-2-yloxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-357

The title compound was prepared according to the schemes, steps andintermediates described below.

A) TBDMSCl, imidazole, CH₂Cl₂, 0° C., 2 h; B) DIAD, PPh₃, Et₃N, dry THF,rt, 1 h; C) H₂, Raney Ni, MeOH, 2 h; D) Pd(OAc)₂, BINAP, Cs₂CO₃,toluene, 110° C., 6 h; E) TFA, CH₂Cl₂, rt, 1 h; F) (BOC)₂O, 30 min, thenK₂CO₃, NMP, 0° C., 15 min.

Step-1

To stirred solution of 1 (1 g, 13.1 mmol) in DCM was added at 0° C.,imidazole (0.875 g, 13.1 mmol) and tert-butyldimethylsilyl chloride(1.98 g, 13.1 mmol). The same temperature was maintained for 2 h, andthen the reaction mixture was filtered and concentrated. The residue waspurified by column chromatography (neutral alumina, pet ether/ethylacetate 7/3) to give 2 (1.4 g, 56%) as a colorless liquid.

Step-2

To a stirred solution of 2 (1.5 g, 7.89 mmol) in THF (15 mL) were added3 (1.36 g, 7.89 mmol), PPh₃ (2.27 g, 8.6 mmol) and Et₃N (1.19 g, 11.1mmol) under N₂ atmosphere. The reaction mixture was cooled to 0° C. andto it was added DIAD (1.75 g, 8.6 mmol). The reaction mixture wasallowed to come to rt and stirred at it for 1 h. It was quenched withwater, extracted with ethyl acetate (3×5 mL) and the combined EtOAcextract was washed with water and brine solution (5 mL each). Theresidue obtained after concentration under reduced pressure was purifiedby column chromatography (SiO₂, 60-120, pet ether/ethyl acetate, 7/3) togive 4 (2.1 g, 76.9%) as a yellow oil.

Step-3

To a solution of 4 (2 g, 5.7 mmol) in methanol (20 mL)) was added RaneyNi (3 g). The reaction mixture was allowed to stir under H₂ atmosphere(bladder pressure) at room temperature for 2 h. The reaction mixture wasfiltered through a pad of Celite® and concentrated under reducedpressure and the residue was purified by column chromatography (neutralalumina, pet ether/ethyl acetate, 8/2) to give 5 (1.4 g, 77%) as abrownish viscous oil.

Step-4

A solution of 6 (0.2 g, 0.63 mmol), prepared according to Step-1 ofExample 20, 1 (0.213. g, 0.63 mmol), Pd(OAc)₂ (0.014 g, 0.063 mmol),BINAP (0.0019 g, 0.031 mmol) and Cs₂CO₃ (0.511 g, 1.5 mmol) in degassedtoluene (toluene was purged with N₂ for 30 min) was heated at 110° C.for 16 h under N₂ atmosphere. The reaction mixture was cooled, dilutedwith EtOAc (20 mL), washed with water (10 mL), brine (10 mL) and driedover Na₂SO₄. Filtration followed by concentration under reduced pressureoffered a residue which was further purified using column chromatography(SiO₂, 60-120, pet ether/ethyl acetate 7/3) to give 7 (0.15 g, 38.4%) asa yellow solid.

Step-5

To a stirred solution of 7 (0.15 g, 0.24 mmol) in dry CH₂Cl₂ (5 mL) at0° C. was added CF₃COOH (1.5 mL) and the reaction mixture was stirred at0° C. for 30 min. The reaction was allowed to come to rt and stirred atit for 1 h. It was concentrated under reduced pressure and the residuewas quenched with NaHCO₃ solution (3 mL) and extracted with EtOAc (3×25mL). The combined EtOAc extract was washed with water (20 mL), brine (10mL) and dried over Na₂SO₄ and concentrated under reduced pressure togive 8 (0.085 g, 86.7%) as a white solid.

Step-6

A stirred solution of 8 (0.085 g, 0.21 mmol) in NMP (2.0 mL) was cooledto 0° C. and to it was added K₂CO₃ (0.29 g, 2.1 mmol) and acryloylchloride (1 M solution in THF, 0.21 mL, 0.21 mmol) and the reactionmixture was stirred at 0° C. for 30 min. The reaction mixture was addeddropwise to a cold, stirring solution of 10% NaHCO₃. After the additionwas over, the solution was stirred for another 30 min at 0° C., and thesolid was isolated by filtration through a Buchner funnel. The solid waswashed with cold water, hexane and was dissolved inmethanol:dichloromethane (50:50, 25 mL) and concentrated under reducedpressure. The residue obtained was suspended in cold water (3 mL), Et₃Nwas added to it and it was extracted with ethyl acetate (2×5 mL). Thecombined ethyl acetate extract was washed with water (5 mL), brine (5mL), dried over Na₂SO₄ and concentrated under reduced pressure to givethe title compound (65 mg, 67%) as yellow solid. ¹H NMR (DMSO-d6) δ ppm:1.18 (d, J=6.12 Hz, 3H), 3.40-3.47 (m, 1H), 3.50-3.56 (m, 1H), 4.20-4.30(m, 1H), 4.82 (t, J=5.6 Hz, 1H), 5.75 (dd, J=1.88 & 10.08 Hz, 1H), 6.25(dd, J=1.92 & 16.92 Hz, 1H), 6.45 (dd, J=10.08 & 16.92 Hz, 1H), 7.12 (d,J 8.76 Hz, 1H), 7.29 (t, J=8.08 Hz, 1H), 7.40-7.44 (m, 3H), 7.52 (d,J=8.44 Hz, 1H), 7.91 (s, 1H), 8.12 (d, J=3.64 Hz, 1H), 9.21 (s, 1H),9.45 (s, 1H), 10.12 (s, 1H); LCMS: m/e 458.0 (M+1).

Example 249 Preparation of(R)—N-(3-(2-(4-chloro-3-(1-hydroxypropan-2-yloxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-358

The title compound was prepared according to the schemes, steps andintermediates described in Example 248 by using (R)-propane-1,2-diol inplace of 1 in Step-1. ¹H NMR (DMSO-d6) δ ppm: 1.18 (d, J=6.12 Hz, 3H),3.40-3.47 (m, 1H), 3.50-3.56 (m, 1H), 4.20-4.30 (m, 1H), 4.82 (t, J=5.6Hz, 1H), 5.75 (dd, J=1.88 & 10.08 Hz, 1H), 6.25 (dd, J=1.92 & 16.92 Hz,1H), 6.45 (dd, J=10.08 & 16.92 Hz, 1H), 7.12 (d, J=8.76 Hz, 1H), 7.29(t, J=8.08 Hz, 1H), 7.40-7.44 (m, 3H), 7.52 (d, J=8.44 Hz, 1H), 7.91 (s,1H), 8.12 (d, J=3.64 Hz, 1H), 9.21 (s, 1H), 9.45 (s, 1H), 10.12 (s, 1H);LCMS: m/e 458.0 (M+1).

Example 250 Preparation of(E)-4-(dimethylamino)-N-(3-(5-methyl-4-(m-tolylamino)pyrimidin-2-ylamino)phenyl)but-2-enamideI-360

The title compound was prepared according to the schemes, steps andintermediates described in Example 3 by using(E)-4-(dimethylamino)but-2-enoyl chloride place of acryloyl chloride inStep-3. ¹H NMR (DMSO-d6) δ ppm: 7.91 (s, 1H), 7.85 (s, 1H), 7.52 (d,J=6.4 Hz, 1H), 7.45 (d, J=7.8 Hz, 1H), 7.34 (s, 1H), 7.31-7.26 (m, 1H),7.21 (dd, J=8.2, 8.0 Hz, 1H), 7.01-6.92 (m, 4H); 6.27 (s, 1H), 6.06 (d,J=15.1 Hz, 1H), 3.14 (d, J=5.5 Hz, 2H), 2.37 (s, 3H), 2.31 (s, 6H), 2.13(s, 3H); LCMS m/z 417 (M+1).

Example 251 Preparation ofN-(3-(2-(3,4-bis(2-methoxyethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-277

The title compound was prepared according to the schemes, steps andintermediates described below.

A) K₂CO₃, Cs₂CO₃, DMF, 90° C., 16 h; B) HNO₃, Acetic acid, r.t., 1 h;

C) Pd/C, methanol, r.t., 3 h; D) Acetic acid, ethanol, 80° C., 16 h;

E) Pd/C, methanol, r.t., 16 h; F) Acryloyl chloride, K₂CO₃, NMP, r.t.,30 min.

Step-1

To a stirred solution of 1 (5 g, 45.4 mmol) in DMF (100 mL) were addedK₂CO₃ (15.6 g, 112.9 mmol), Cs₂CO₃ (36.6 g, 112.3 mmol) followed by 2(12.4 mL, 135.8 mmol) and the reaction mixture was heated to 90° C. for16 h. Then the reaction mixture was cooled, filtered and the filtratewas concentrated under reduced pressure. The residue was diluted withethyl acetate, washed with water and brine, dried over anhydrous Na₂SO₄,filtered and concentrated to give 3 (9.9 g, 96%) as brown liquid.

Step-2

To a stirred solution of 3 (2.5 g, 11 mmol) in acetic acid (50 mL) wasadded fuming nitric acid (0.83 g, 13.2 mmol) at 0° C. drop wise andstirred at room temperature for 1 h. The reaction mixture was pouredonto crushed ice and the solid obtained was collected by filtration anddried under vacuum to give 4 (2.23 g, 74%) as yellow solid.

Step-3

To a solution of 4 (1 g, 3.68 mmol) in methanol (20 mL) was added Pd/C(0.2 g, 20% w/w) under N₂ atmosphere. Then the reaction mixture wasstirred under H₂ pressure (bladder) for 3 h. It was filtered throughcelite bed and the filtrate was concentrated to give 5 (0.87 g, 98%) asbrown oil.

Step-4

To a solution of 6 (0.45 g, 1.675 mmol) in ethanol (4 mL) were added 5(0.4 g, 1.658 mmol) and acetic acid (0.2 ml) and the reaction mixturewas heated to 80° C. for 16 h. Then it was concentrated under vacuum andthe residue was purified by column chromatography (SiO₂, petroleumether:ethyl acetate, 6:4) to get 7 (0.13 g, 17%) as yellow solid.

Step-5

To a solution of 7 (0.13 g, 0.275 mmol) in methanol (3 mL) was addedPd/C (26 mg, 20% w/w) under N₂ atmosphere. Then the reaction mixture wasstirred at room temperature under H₂ pressure (bladder) for 16 h. It wasfiltered through celite bed and the filtrate was concentrated to give 8(0.10 g, 82%) as green solid.

Step-6

To a cooled solution of 8 (0.1 g, 0.225 mmol) in NMP (1.0 mL) was addedK₂CO₃ (0.15 g, 1.1 mmol), acryloyl chloride (0.002 g, 0.25 mmol) and thereaction mixture was stirred at 0° C. for 30 min. Then the reactionmixture added drop-wise to a cold, stirring solution of 10% NaHCO₃ (˜5ml) and stirred at 0° C. for 30 min. A solid precipitated out wasisolated by filtration through a Buchner funnel. The solid was dissolvedin methanol:dichloromethane (1:1, 25 mL) and concentrated under reducedpressure. The residue obtained was suspended in cold water (3 mL), Et₃Nwas added to it and it was extracted with ethyl acetate. The combinedethyl acetate extract was washed with water and brine, dried over Na₂SO₄and concentrated under reduced pressure to get I-277 (0.049 g, 44%) asyellow solid.

¹H NMR (DMSO-d₆): δ=3.28 (s, 3H), 3.29 (s, 3H), 3.56-3.60 (m, 4H), 3.88(t, J=4.8 Hz, 2H), 3.98 (t, J=4.9 Hz, 2H), 5.74 (d, J=12.1 Hz, 1H), 6.24(d, J=14 Hz, 1H), 6.45 (dd, J=16, 10 Hz, 1H), 6.75 (d, J=8.7 Hz, 1H),7.21-727 (m, 3H), 7.41 (d, J=8 Hz, 1H), 7.52 (d, J=8 Hz, 1H), 7.89 (s,1H), 8.07 (d, J=3.7 Hz, 1H), 8.95 (s, 1H), 9.38 (s, 1H), 10.12 (s, 1H);LCMS: m/e: 498.2 (M+1).

Example 252 Preparation of tert-butyl2-(2-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)phenoxy)ethoxy)ethylcarbamateI-370

The title compound was prepared according to the schemes, steps andintermediates described in Example 245, by using tert-butyl2-(2-(4-aminophenoxy)ethoxy)ethylcarbamate in the place of 2 in Step 1.¹H NMR (DMSO-d₆): δ=1.37 (s, 9H), 3.09 (m, 2H), 3.43 (t, J=6.0 Hz, 2H),3.68 (d, J=4.3 Hz, 2H), 3.98 (t, J=4.3 Hz, 2H), 5.75 (d, J=11.4 Hz, 1H),6.25 (d, J=15.6 Hz, 1H), 6.45 (dd, J=16.9, 10 Hz, 1H), 6.73-6.81 (m,3H), 7.27 (t, J=8.1 Hz, 1H), 7.40 (d, J=7.9 Hz, 1H), 7.47-7.53 (m, 3H),7.92 (s, 1H), 8.06 (d, J=3.6 Hz, 1H), 8.96 (s, 1H), 9.36 (s, 1H), 10.12(s, 1H); LCMS: m/e: 553.2 (M+1).

Example 253 Preparation ofN1-(2-(2-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)phenoxy)ethoxy)ethyl)-N5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramideI-363

The title compound was prepared according to the schemes, steps andintermediates described in Example 204, by using tert-butyl2-(2-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)phenoxy)ethoxy)ethylcarbamate(I-370, described in Example 252) in the place of I-45 in Step 1. LC/MS(RT=2.045/(M+H)) 995.4.

Example 254 Preparation of(R)—N-(3-(5-fluoro-2-(3-fluoro-4-(1-hydroxypropan-2-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-372

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(R)-2-(4-amino-2-fluorophenoxy)propan-1-ol in the place of 4 in Step 2.¹HNMR (DMSO-d₆): δ=1.17 (d, J=6.2 Hz, 3H), 3.40-3.46 (m, 1H), 3.50-3.56(m, 1H), 4.22 (q, J=5.9 Hz, 1H), 4.83 (t, J=5.8 Hz, 1H), 5.75 (dd,J=10.1, 1.9 Hz, 1H), 6.25 (dd, J=16.9, 1.9 Hz, 1H), 6.46 (dd, J=16.9,10.1 Hz, 1H), 6.98 (t, J=9.4 Hz, 1H), 7.24-7.31 (m, 2H), 7.42 (d, J=8.2Hz, 1H), 7.49 (d, J=7.9 Hz, 1H), 7.68 (dd, J=14.3, 2.4 Hz, 1H), 7.93 (s,1H), 8.11 (d, J=3.7 Hz, 1H), 9.20 (s, 1H), 9.45 (s, 1H), 10.14 (s, 1H);LCMS: m/e 442.2 (M+1).

Example 255 Preparation of(S)—N-(3-(5-fluoro-2-(3-fluoro-4-(1-hydroxypropan-2-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamideI-373

The title compound was prepared according to the schemes, steps andintermediates described in Example 20, by using(S)-2-(4-amino-2-fluorophenoxy)propan-1-ol in the place of 4 in Step 2.LC/MS (RT=3.316/(M+H)) 442.0.

Example 256 Preparation of(S)-((R)-2-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)-2-fluorophenoxy)propyl)2-amino-3-methylbutanoate I-374

The title compound was prepared according to the schemes, steps andintermediates described below.

A) EDC.HCl, HOBT, DMAP, NMM, DMF, r.t., 16 h;

B) HCl (1M in Diethylether), 0° C., 1 h.

Step-1

To a stirred solution of I-372 (0.08 g, 0.18 mmol) in DMF (0.8 mL) wereadded 2 (0.057 g, 0.26 mmol), EDC.HCl (0.086 g, 0.45 mmol), HOBT (0.06g, 0.44 mmol), 4-DMAP (0.008) and NMM (0.036 g, 0.36 mmol). And thereaction mixture was stirred at room temperature for 16 h. Then it wasquenched with water and extracted with ethyl acetate. The ethyl acetatelayer was washed with water and brine, dried over anhydrous Na₂SO₄,filtered and concentrated. The residue was purified by columnchromatography (SiO₂, chloroform:methanol, 9:1) to obtain 3 (0.06 g,51.7%) as off white solid.

Step-2

A cold solution of HCl in diethyl ether (1M, 6 ml) was added to 3 (60mg, 0.09 mmol) and the reaction mixture was stirred at 0° C. for 45 min.Then the reaction mixture was concentrated and the solid obtained waswashed with diethyl ether and dried to yield I-374 (as its HCl salt) asyellow solid (20 mg). 1H NMR (DMSO-d₆) δ=0.77 (d, J=6.8 Hz, 3H), 0.83(d, J=6.8 Hz, 3H), 1.23 (d, J=6.3 Hz, 3H), 1.75-1.83 (m, 1H), 3.10 (d,J=5.2 Hz, 1H), 4.05-4.11 (m, 1H), 4.22 (dd, J=11.7, 3.3 Hz, 1H),4.40-4.50 (m, 1H), 5.73 (d, J=10.2 Hz, 1H), 6.23 (d, J=17.1 Hz, 1H),6.44 (dd, J=17, 10.2 Hz, 1H), 6.98 (t, J=9.2 Hz, 1H), 7.24-7.29 (m, 2H),7.41 (d, J=7.8 Hz, 1H), 7.48 (d, J=7.8 Hz, 1H), 7.68 (d, J=14.3 Hz, 1H),7.91 (s, 1H), 8.10 (d, J=3.5 Hz, 1H), 9.23 (s, 1H), 9.44 (s, 1H), 10.13(s, 1H). LCMS: m/e 541.2 (M+1).

Example 257 Preparation of(S)-((S)-2-(4-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)-2-fluorophenoxy)propyl)2-amino-3-methylbutanoate I-377

The title compound was prepared according to the schemes, steps andintermediates described in Example 256, by using(S)—N-(3-(5-fluoro-2-(3-fluoro-4-(1-hydroxypropan-2-yloxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide(I-373 described in example 255) in the place of I-372 in Step 1. LC/MS(RT=3.227/(M+H)) 542.2.

Example 258 Preparation of(S)-((S)-2-(5-(4-(3-acrylamidophenylamino)-5-fluoropyrimidin-2-ylamino)-2-chlorophenoxy)propyl)2-amino-3-methylbutanoate I-369

The title compound was prepared according to the schemes, steps andintermediates described in Example 256, by using(S)—N-(3-(2-(4-chloro-3-(1-hydroxypropan-2-yloxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamide(I-357 described in example 248) in the place of I-372 in Step 1. LC/MS(RT=3.54/(M+H)) 557.2.

Example 259 Preparation ofN-(3-(2-(4-(2-(2-azidoethoxy)ethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-371

The title compound was prepared according to the schemes, steps andintermediates described below.

A) K₂CO₃, DMSO, r.t., 16 h; B) Pd/C, Ethanol, 16 h; C) Pd(OAc)₂, BINAP,Cs₂CO₃, Toluene, 110° C., 16 h; D) Mesyl chloride, Et₃N, CH₂Cl₂ r.t., 1h; E) Sodium azide, DMF, 70° C., 4 h; F) TFA, CH₂Cl₂, 0° C.-r.t., 1.5 h;G) Acryloyl chloride, NMP, K₂CO₃, 30 min.

Step-1

To a stirred solution of 1 (1 g, 7.09 mmol) in DMSO (20 mL) was addedK₂CO₃ (0.96 g, 6.95 mmol) and stirred at room temperature for 15 min.Then 2 (3.7 g, 34.86 mmol) was added and stirring continued at roomtemperature for 16 h. The reaction mixture was poured into ice water andextracted with ethyl acetate. The organic layer was washed with waterand brine, dried over anhydrous Na₂SO₄, filtered and concentrated toobtain 3 as yellow solid (1.0 g, 62%).

Step-2

To a solution of 3 (1.0 g, 4.4 mmol) in ethanol was added Pd/C (0.1 g,10% w/w) under N₂ and then the reaction mixture was stirred under H₂pressure (bladder) for 16 h. Then the reaction mixture was filtered andthe filtrate was concentrated to obtain 4 as brown viscous liquid (0.8g, 92.2%).

Step-3

A mixture of 4 (0.3 g, 1.52 mmol), 5 (0.61 g, 1.8 mmol), Pd(OAc)₂ (0.017g, 0.076 mmol), BINAP (0.066 g, 0.106 mmol) and Cs₂CO₃ (1.23 g, 3.78mmol) in degassed toluene (20 mL) (toluene was purged with N₂ for 30min) was heated at 110° C. for 16 h under N₂ atmosphere. The reactionmixture was cooled, diluted with EtOAc, washed with water and brine anddried over Na₂SO₄. Filtration followed by concentration under reducedpressure offered a residue which was further purified by columnchromatography (SiO₂, petroleum ether:ethyl acetate; 6:4) to obtain 6(0.3 g, 39.5%) as a brown solid.

Step-4

To a stirred solution of 6 (0.35 g, 0.7 mmol) and triethylamine (0.2 mL,1.4 mmol) in dichloromethane (10 mL) at 0° C. was added mesyl chloride(0.12 g, 1.05 mmol) and then the reaction mixture stirred at roomtemperature for 1 h. It was quenched with water; the organic layer wasseparated, dried over anhydrous Na₂SO₄ and concentrated to obtain 7 ascolorless oil (0.4 g, crude).

Step-5

To a stirred solution of 7 (0.4 g, 0.69 mmol) in DMF (5 mL) was addedsodium azide (0.054 g, 0.83 mmol) and the reaction mixture was heated to70° C. for 4 h. Then it was treated with ice water and extracted withethyl acetate. The ethyl acetate layer was washed with water and withbrine, dried over anhydrous Na₂SO₄, filtered and concentrated to obtain8 as viscous oil 8 (0.3 g, 82.6%).

Step-6

To a stirred solution of 3 (0.3 g, 0.57 mmol) in dry CH₂Cl₂ (10 mL) at0° C. was added CF₃COOH (1.0 mL) and the reaction mixture was stirred at0° C. for 30 min and then at r.t. for 1 h. It was concentrated underreduced pressure and the residue was treated with NaHCO₃ solution andextracted with ethyl acetate. The combined EtOAc extract was washed withwater. Then it was extracted with 10% citric acid solution. The combinedcitric acid extract was basified with 10% NaOH solution and thenextracted with EtOAc. The EtOAc extract was washed with water and brine,dried over Na₂SO₄ and concentrated to get 9 (0.17 g, 70%) as brownsolid.

Step-7

To a stirred solution of 9 (0.075 g, 0.177 mmol) in NMP (1.5 mL) at 0°C. was added K₂CO₃ (0.05 g, 0.36 mmol) followed by acryloyl chloride(0.017 g, 0.188 mmol) and the reaction mixture was stirred at 0° C. for30 min. Then the reaction mixture was added drop wise to a cold stirringsolution of 10% NaHCO₃. After the addition was over, the solution wasstirred for another 30 min at 0° C. and the solid separated was isolatedby filtration through a Buchner funnel. The solid was washed with coldwater, hexane and was dissolved in methanol:dichloromethane (1:1, 5 mL)and then concentrated under reduced pressure. The residue obtained wassuspended in cold water (3 mL) and Et₃N (˜1 ml), stirred for a while andextracted with ethyl acetate. The combined ethyl acetate extract waswashed with water and brine, dried over Na₂SO₄ and concentrated underreduced pressure. The residue obtained was purified by columnchromatography twice (SiO₂, petroleum ether:ethyl acetate, 1:1) to getI-371 (0.04 g, 47.3%) as white solid. ¹H NMR (DMSO-d₆): δ=3.42 (t, J=4.9Hz, 1H), 3.66 (t, J=4.9 Hz, 1H), 3.72-3.77 (m, 4H), 4.00 (t, J=4.6 Hz,2H), 5.75 (dd, J=10.1, 1.8 Hz, 1H), 6.25 (dd, J=17, 1.9 Hz, 1H), 6.45(dd, J=16.9, 10.1 Hz, 1H), 6.75 (d, J=9 Hz, 2H), 7.27 (t, J=8.1 Hz, 1H),7.40 (d, J=8 Hz, 1H), 7.47-7.53 (m, 3H), 7.92 (s, 1H), 8.06 (d, J=3.7Hz, 1H), 8.97 (s, 1H), 9.37 (s, 1H), 10.12 (s, 1H); LCMS: m/e 479.2(M+1).

Example 260 Preparation ofN-(3-(2-(4-(2-azidoethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-376

The title compound was prepared according to the schemes, steps andintermediates described in Example 259, by using2-(4-aminophenoxy)ethanol in the place of 4 in Step 3. LC/MS(RT=3.447/(M+H)) 435.2.

Example 261 Preparation ofN-(3-(2-(4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)acrylamideI-379

The title compound was prepared according to the schemes, steps andintermediates described in Example 259, by using2,2′-(ethane-1,2-diylbis(oxy))diethanol in the place of 2 in Step 1.LC/MS (RT=4.454/(M+H))475.6.

Example 262 Preparation of(E)-N-(3-(2-(4-chloro-3-(2-methoxyethoxy)phenylamino)-5-fluoropyrimidin-4-ylamino)phenyl)but-2-enamideI-375

The title compound was prepared according to the schemes, steps andintermediates described in Example 238, by using (E)-but-2-enoylchloride in the place of 7 in Step 4. LC/MS (RT=4.026/(M+H)) 473.2.

Example 263 Preparation ofN-(3-(5-fluoro-4-(4-(2-methoxyethoxy)phenylamino)pyrimidin-2-ylamino)phenyl)acrylamideI-381

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using4-(2-methoxyethoxy)aniline in the place of 2 in Step 1. LC/MS(RT=2.275/(M+H)) 424.1.

Example 264 Preparation ofN-(3-(5-fluoro-4-(3-(2-morpholinoethoxy)phenylamino)pyrimidin-2-ylamino)phenyl)acrylamideI-382

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using3-(2-morpholinoethoxy)aniline in the place of 2 in Step 1. LC/MS(RT=2.108/(M+H)) 479.2.

Example 265 Preparation of(S)—N-(3-(5-fluoro-4-(4-(tetrahydrofuran-3-yloxy)phenylamino)pyrimidin-2-ylamino)phenyl)acrylamideI-338

The title compound was prepared according to the schemes, steps andintermediates described in Example 4, by using(S)-4-(tetrahydrofuran-3-yloxy)aniline in the place of 2 in Step 1.LC/MS (RT=1.71/(M+H)) 458.1.

Biological Examples

Described below are assays used to measure the biological activity ofprovided compounds as inhibitors of BTK, TEC, ITK, BMX, ErbB1 (EGFR),ErbB2, ErbB4, and JAK3.

Example 266

Omnia Assay Protocol for Potency Assessment Against BTK

Below describes the protocol using EGFR-WT and EGFR-T790M/L858R and theprotocol BTK-optimized reagent conditions then follow.

The mechanics of the assay platform are best described by the vendor(Invitrogen, Carlsbad, Calif.) on their website at the following URL:www.invitrogen.com/content.cfm?pageid=11338 orwww.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Drug-Discovery/Target-and-Lead-Identification-and-Validation/KinaseBiology/KB-Misc/Biochemical-Assays/Omnia-Kinase-Assays.html.

Briefly, 10× stocks of EGFR-WT (PV3872) from Invitrogen andEGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif.,1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates(KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mMTris, pH 7.5, 5 mM MgCl₂, 1 mM EGTA, 5 mM β-glycerophosphate, 5%glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of eachenzyme were pre-incubated in a Corning (#3574) 384-well, white,non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27°C. with a 0.5 μL volume of 50% DMSO and serially diluted compoundsprepared in 50% DMSO. Kinase reactions were started with the addition of45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90seconds for 60 minutes at λ_(ex)360/λ_(em)485 in a Synergy⁴ plate readerfrom BioTek (Winooski, Vt.). At the conclusion of each assay, progresscurves from each well were examined for linear reaction kinetics and fitstatistics (R², 95% confidence interval, absolute sum of squares).Initial velocity (0 minutes to ˜30 minutes) from each reaction wasdetermined from the slope of a plot of relative fluorescence units vstime (minutes) and then plotted against inhibitor concentration toestimate IC₅₀ from log[Inhibitor] vs Response, Variable Slope model inGraphPad Prism from GraphPad Software (San Diego, Calif.).

The modified BTK-optimized reagent conditions for the above protocolare:

-   -   [BTK]=5 nM, [ATP]=40 mM, [Y5-Sox]=10 mM (ATP KMapp˜36 mM).

Example 267

Table 6 shows the activity of selected compounds of this invention inthe BTK inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≤10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≥10,000 nM.

TABLE 6 BTK Inhibition Data Compound # BTK Inhibition I-1 A I-2 A I-3 AI-4 A I-5 A I-7 A I^(R)-7 C I-8 A I-9 A I-10 C I-11 A I-23 B I-27 A I-28A I-33 A I-34 B I-35 A I-38 A I-39 A I-40 A I-45 A I-54 A I-55 A I-56 CI-60 C I-69 A I-70 A I-71 A I-72 A I-73 A I-74 A I-75 A I-76 A I-77 AI-78 A I-79 A I-80 A I-81 A I-82 A I-83 A I-84 A I-85 B I-86 A I-87 AI-88 A I-89 A I-90 A I-91 A I-92 A I-93 A I-94 A I-95 A I-96 A I-97 AI-98 C I-99 A I-100 C I-101 B I-102 B I-103 A I-104 A I-105 A I-106 BI-107 A I-108 A I-109 A I-110 A I-111 A I-112 A I-113 A I-114 A I-115 BI-116 A I-117 A I-118 A I-119 B I-120 A I-121 A I-122 A I-123 B I-124 AI-125 A I-126 A I-127 B I-128 A I-129 A I-130 A I-131 A I-132 A I-133 AI-134 A I-135 A I-136 A I-137 C I-138 A I-139 A I-140 A I-141 A I-142 AI-143 A I-144 A I-145 A I-146 A I-147 A I-148 D I-149 A I-150 A I-151 AI-152 A I-153 A I-154 A I-155 A I-156 A I-157 A I-158 A I-159 A I-160 AI-161 A I-162 A I-163 B I-164 A I-165 A I-166 A I-167 A I-168 A I-169 AI-170 A I-171 A I-172 A I-173 A I-174 A I-175 C I-176 A I-177 A I-178 AI-179 C I-180 A I-181 A I-182 A I-183 A I-184 A I-185 A I-186 A I-187 BI-188 A I-189 A I-190 A I-191 A I-192 A I-193 C I-194 A I-195 A I-196 BI-197 C I-198 A I-199 A I-200 A I-201 A I-202 A I-203 B I-204 A I-205 BI-206 E I-207 A I-208 A I-209 A I-210 A I-211 D I-212 D I-213 E I-214 BI-215 A I-216 C I-217 A I-218 A I-219 A I-220 A I-221 B I-222 B I-223 EI-224 B I-225 C I-226 B I-227 A I-228 A I-229 B I-230 A I-231 C I-232 BI-233 A I-234 D I-235 B I-236 B I-237 A I-238 D I-241 D I-242 A I-243 AI-244 A I-245 A I-246 B I-247 A I-248 A I-249 A I-277 A I-312 A I-313 AI-315 A I-316 A I-318 A I-321 A I-322 A I-323 C I-324 A I-325 C I-326 AI-327 A I-328 A I-329 A I-330 B I-331 B I-332 A I-333 A I-334 A I-335 AI-336 A I-337 A I-338 A I-339 A I-341 A I-342 A I-343 B I-344 A I-345 AI-346 A I-347 A I-348 A I-349 A I-350 A I-351 A I-352 A I-353 A I-354 AI-355 A I-356 A I-357 A I-358 A I-359 A I-360 A I-362 A I-363 A I-369 AI-370 A I-371 A I-372 A I-373 A I-374 A I-375 D I-376 A I-377 A I-378 AI-379 A I-381 A I-382 B

Example 268

BTK Ramos Cellular Assay

Compounds I-2, I-4, and I-7 were assayed in Ramos human Burkitt lymphomacells. Ramos cells were grown in suspension in T225 flasks, spun down,resuspended in 50 ml serum-free media and incubated for 1 hour. Compoundwas added to Ramos cells in serum free media to a final concentration of1, 0.1, 0.01, or 0.001 μM. Ramos cells were incubated with compound for1 hour, washed again and resuspended in 100 ul serum-free media. Cellswere then stimulated with 1 μg of goat F(ab′)2 Anti-Human IgM andincubated on ice for 10 minutes to activate B cell receptor signalingpathways. After 10 minutes, the cells were washed once with PBS and thenlysed on ice with Invitrogen Cell Extraction buffer. 16 g total proteinfrom lysates were loaded on gel and blots were probed forphosphorylation of the BTK substrate PLCγ2. Dose response inhibition ofBTK signaling in Ramos cells is depicted in FIGS. 1, 2, 3, 4 and 5.

Table 7 shows the activity of selected compounds of this invention inthe BTK Ramos cellular inhibition assay. The compound numbers correspondto the compound numbers in Table 5. Compounds having an activitydesignated as “A” provided an IC₅₀≤10 nM; compounds having an activitydesignated as “B” provided an IC₅₀ 10-100 nM; compounds having anactivity designated as “C” provided an IC₅₀ of 100-1000 nM; compoundshaving an activity designated as “D” provided an IC₅₀ of 1000-10,000 nM;and compounds having an activity designated as “E” provided anIC₅₀≥10,000 nM.

TABLE 7 BTK Ramos Cellular Inhibition Data Compound # BTK Inhibition I-3B I-4 B I-7 A I-8 C I-27 B I-33 A I-35 A I-38 B I-39 B I-40 B I-45 AI-77 A I-78 A I-79 A I-80 A I-86 A I-87 A I-95 A I-96 A I-97 A I-103 AI-105 A I-107 B I-108 B I-110 B I-114 B I-116 A I-118 A I-121 B I-122 AI-124 A I-125 B I-126 B I-128 B I-129 A I-131 A I-133 B I-134 B I-135 BI-138 A I-139 B I-140 B I-142 B I-143 A I-147 B I-149 B I-150 B I-151 AI-152 B I-153 A I-154 A I-156 A I-157 A I-158 B I-159 C I-160 B I-162 AI-163 B I-164 A I-165 B I-166 B I-167 B I-168 B I-169 A I-170 B I-172 AI-173 A I-174 A I-176 C I-177 C I-178 B I-180 C I-182 A I-185 C I-186 AI-188 A I-189 B I-190 B I-192 B I-194 A I-195 B I-198 A I-201 C I-202 CI-204 A I-207 C I-208 B I-209 B I-210 A I-217 A I-219 B I-220 B I-227 AI-228 A I-230 A I-233 A I-242 A I-243 A I-244 B I-245 A I-247 A I-248 AI-249 A I-313 A I-315 A I-316 A I-318 A I-321 A

Example 269

Washout Experiment with Ramos Cells

Ramos cells were serum starved for one hour in RPMI media+1% glutamineat 37° C. After starvation, Ramos cells were treated with 100 nMcompound diluted in serum free RPMI media for 1 hour. After compoundtreatment, the media was removed and cells were washed withcompound-free media. Subsequently, Ramos cells were washed every 2 hoursand resuspended in fresh compound-free media. Cells were collected atspecified timepoints, treated with 1 ug anti-human IgM (Southern Biotechcat #2022-01) for 10 minutes on ice to induce BCR signaling and thenwashed in PBS. Ramos cells were then lysed in Cell Extraction Buffer(Invitrogen FNN0011) supplemented with Roche complete protease inhibitortablets (Roche 11697498001) and phosphatase inhibitors (Roche 04 906 837001) and 18 ug total protein lysate was loaded in each lane. Inhibitionof BTK kinase activity was assayed by measuring its substrate (PLCγ2)phosphorylation by western blot with phospho-specific antibodies fromCell Signaling Technologies cat#3871. The results of this experimentwith compounds I-2, I-4 and I-7 are depicted in FIGS. 1, 2 and 3.

Table 8 provides data for selected compounds in the Ramos washout assay.

TABLE 8 BTK Washout Data Compound # BTK Inhibition Type I-2 irreversibleI-4 irreversible I-7 irreversible I-28 irreversible I-35 irreversibleI-38 reversible I-228 irreversible I-230 irreversible I-242 irreversibleI-243 irreversible I-247 irreversible I-248 irreversible

Example 270

Mass Spectrometry for BTK

Intact BTK was incubated for 1 hr at a 10× fold excess of I-7 toprotein. Aliquots (2 μl) of the samples were diluted with 10 μl of 0.1%TFA prior to micro C4 ZipTipping directly onto the MALDI target usingSinapinic acid as the desorption matrix (10 mg/ml in 0.1%TFA:Acetonitrile 20:80). See FIG. 15. Top panel shows the mass spectrace of the intact BTK protein (m/z 81.032 Da). The bottom panel showsmass spec trace when BTK was incubated with I-7 (mw=345.4). The centroidmass (m/z=81.403 Da) shows a positive shift of about 371.1 Da indicatingcomplete modification of BTK by I-7. Other compounds that completelymodify BTK include I-96, I-71, I-149, I-161, I-163, I-182, I-195, I-207,I-219, and I-244.

Example 271

Human Primary B Cell Proliferation Assay

Human naïve B cells were purified from 100 mL whole blood using a MACSpurification kit designed to isolate CD19+, IgD+ cells by negativeselection. Purified naïve B cells were resuspended in RPMI complete andstimulated with 5 μg/ml α-IgM for 72 hours. ³H-Thymidine was included inthe culture media for the final 16 h, cells were harvested and ³Hincorporation measured. Inhibition of B cell proliferation correlateswith inhibition of BTK substrate phosphorylation after α-IgMstimulation. Importantly, a molecule with the same scaffold as I-7 butbiochemically inactive against BTK, I^(R)-7, is not active in the naïveB cell proliferation assay.

TABLE 9 Compound # EC₅₀ (nM) I-7 1-10 I^(R)-7 >1000 I-190 10-100 I-1821-10 I-96 1-10

Example 272

B Cell Lymphoma Proliferation Assay

Provided compounds inhibit proliferation of various B cell lymphoma celllines, as shown in Table 10. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an EC₅₀<0.1 μM; compounds having an activity designated as“B” provided an EC₅₀ 0.1-1 μM; compounds having an activity designatedas “C” provided an EC₅₀ of 1-10 μM; and compounds having an activitydesignated as “D” provided an EC₅₀ of >10 μM.

TABLE 10 EC50 (μM) Compound # DOHH2 WSU-DLCL2 DHL4 I-2 B — — I-3 B — —I-4 B B B I-7 C C C I-27 C D — I-28 C C — I-35 C C — I-38 C C — I-39 C C— I-40 B C — I-77 B — — I-78 B — — I-79 C — — I-80 B — — I-81 B — — I-86D — — I-87 C — — I-88 C — — I-89 C — — I-90 B — — I-91 C C — I-96 B CI-103 — C — I-105 B C — I-116 D D — I-121 C — — I-122 C — — I-124 C — —I-126 A — — I-128 C — — I-129 C — — I-132 C — C I-133 B — A I-134 B — AI-135 D — D I-138 C — C I-141 C — — I-142 C — — I-143 C C C I-147 C — —I-149 C — — I-150 C C C I-151 C — C I-152 C — — I-153 C C C I-154 C C CI-155 C C C I-156 B C C I-157 C C C I-158 C C C I-159 C — — I-160 C C CI-161 C — C I-162 C C C I-163 C C C I-164 C C C I-165 C C C I-166 C C CI-167 C — — I-168 C — — I-169 C — — I-170 C — — I-171 C — — I-172 C — —I-173 C — — I-174 C — — I-178 C D D I-182 C C C I-184 C — — I-186 C — CI-188 C — — I-189 D D C I-190 D — — I-192 C D — I-194 C C C I-195 C D DI-204 C C — I-207 B C — I-208 C C — I-209 D C — I-210 C C — I-217 D D —I-227 C C — I-228 D D — I-230 C C — I-242 C C — I-243 C C — I-244 B C CI-245 C D — I-247 B C — I-248 C C — I-298 — C —

Example 273

In Vivo Thymus-Independent (TI-2) B Cell Activation

C57/B6 mice were dosed daily with 100 mg/kg of the appropriate compoundon day 0 through 5. Mice were immunized once with 25 μg TNP-Ficoll onday 1, serum was collected on day 6 and analyzed for circulating α-TNPIgM (1:1600 serum dilution) and IgG3 (1:200 serum dilution) antibodyproduction by ELISA. Results represent the average of 10 mice pertreatment group and are given in Table 11 as % inhibition of TI-2independent B cell activation.

TABLE 11 % Inhibition Compound # IgM (1:1600) IgG3 (1:100) I-7 48 57I-182 25 40 I-96 43 37.2

Example 274

Collagen Antibody Induced Arthritis Model

On day 0 baseline footpad measurements were made and animals weredistributed to the experimental groups in such a way as to generategroups with no significant differences between the groups. Each animalwas then inoculated intravenously with 2 mg Arthritomab monoclonalantibody cocktail. Treatment with test agents began at this time. On day6, each animal was injected intraperitoneally with 50 μg LPS in 200 μlof sterile PBS. Footpad measurements and clinical scoring were conductedon days 6, 7, 8, 9, 10, 11, 12, 14, 18, and 21. Table 12 shows theresults.

TABLE 12 % inhibition foot Compound # Dose pad swelling I-7 30 mg/kg 83

Example 275

PG-PS Arthritis Model

On day 0, female Lewis rats received an intraperitoneal (IP) bolus ofpeptidoglycan-polysaccharide (PG-PS) in an amount of 15 g/g rat bodyweight. Baseline control rats received an IP bolus of PBS. The vehicleand treatment groups were dosed via oral gavage just prior to PG-PSadministration. Treatment with vehicle and compound continued each daythrough day 22. Maximal lateral ankle width measurements of both rearlimbs were collected with a caliper throughout the study. On day 23,study was terminated and the final change in ankle swelling wascalculated and compared to vehicle controls. Table 13 shows the resultsfor two compounds (n=number of experiments).

TABLE 13 % inhibition ankle Compound # n swelling I-7 1 77.5 I-96 2 82.8

Example 276

Mass Spectrometry for TEC Kinase (Compound I-2)

TEC kinase (45 pmols; Invitrogen) was incubated with (I-2) (450 pmols)for 3 hrs at 10× excess prior to tryptic digestion. Iodoacetamide wasused as the alkylating agent after compound incubation. A control sample(45 pmols) was also prepared which did not have the addition of (I-2).For tryptic digests a 5 ul aliquot (7.5 pmols) was diluted with 15 ul of0.1% TFA prior to micro C18 Zip Tipping directly onto the MALDI targetusing alpha cyano-4-hydroxy cinnamic acid as the matrix (5 mg/ml in 0.1%TFA:Acetonitrile 50:50).

As depicted in FIG. 6, the expected peptide (GCLLNFLR) to be modifiedwas immediately evident after reaction with 1-2 (MI mass of 359.17 Da)at MH+ of 1294.72. The peptide was also quite evident in the controlsample as modified by lodacetamide at MH+ of 992.56. Interestingly theiodoacetamide modified peptide was not evident in the digest reactedwith compound I-2 indicating that the reaction was complete. There wasno evidence of other modified peptides.

Evidence of compound I-2 was observed at MH+ of 360.17 in the low massrange of the spectra. The fragmentation spectra of the 360.17 peak didshow diagnostic fragments that were apparent in the PSD spectra of themodified peptide at 1294.72 (See FIG. 6).

To further verify the presence of the modified peptides, both theiodoacetamide labeled (992.56) and I-2 labeled (1294.72) were subjectedto PSD (MS/MS) analysis. After a database search of the NCBI nr Homosapien database using Mascot MS/MS Ion Search program the top match wasthe expected peptide in both cases.

Instrumental:

For tryptic digests the instrument was set in Reflectron mode with apulsed extraction setting of 2200. Calibration was done using the LaserBiolabs Pep Mix standard (1046.54, 1296.69, 1672.92, 2093.09, 2465.20).For CID/PSD analysis the peptide was selected using cursors to set iongate timing and fragmentation occurred at a laser power about 20% higherand He was used as the collision gas for CID. Calibration for fragmentswas done using the P14R fragmentation calibration for the Curved fieldReflectron.

Example 277

Mass Spectrometry for TEC Kinase (Compound I-4)

TEC kinase (45 pmols; Invitrogen) was incubated with (I-4) (450 pmols)for 3 hrs at 10× excess prior to tryptic digestion. Iodoacetamide wasused as the alkylating agent after compound incubation. A control sample(45 pmols) was also prepared which did not have the addition of (I-4).For tryptic digests a 5 ul aliquot (7.5 pmols) was diluted with 15 ul of0.1% TFA prior to micro C18 Zip Tipping directly onto the MALDI targetusing alpha cyano-4-hydroxy cinnamic acid as the matrix (5 mg/ml in 0.1%TFA:Acetonitrile 50:50).

As depicted in FIG. 7, the expected peptide (GCLLNFLR) to be modifiedwas immediately evident at MH+ of 1355.72. This is the mass to beexpected when compound I-4, with an adduct mass of 420.21, is added tothe peptide mass of 935.51. The peptide was also quite evident in thecontrol sample as modified by lodacetamide at MH⁺ of 992.56.Interestingly the iodoacetamide modified peptide was not evident in thedigest reacted with compound I-4 indicating that the reaction wascomplete. There was no evidence of other modified peptides.

Evidence of compound I-4 was observed at MH+ of 421.35 in the low massrange of the spectra. The fragmentation spectra of the 421.35 peak didreveal two prominent peaks that were apparent in the PSD spectra of themodified peptide at 1355.72 (See FIG. 7).

To further verify the presence of the modified peptide with compoundI-4, the peptide at MH+ of 1355.72 was subjected to PSD (MS/MS)analysis. Because of the low intensity of fragments, a databasecorrelation was not possible. However, diagnostic fragments from the I-4molecule itself provided confidence in the identification. Diagnosticfragments at MH+ of 376.38 and 421.83 are from I-4.

Instrumental:

For tryptic digests the instrument was set in Reflectron mode with apulsed extraction setting of 1800. Calibration was done using the LaserBiolabs Pep Mix standard (1046.54, 1296.69, 1672.92, 2093.09, 2465.20).For CID/PSD analysis the peptide was selected using cursors to set iongate timing and fragmentation occurred at a laser power about 20% higherand He was used as the collision gas for CID. Calibration for fragmentswas done using the P14R fragmentation calibration for the Curved fieldReflectron.

Example 278

Mass Spectrometry for TEC Kinase (Compound I-7)

TEC kinase (45 pmols; Invitrogen) was incubated with (I-7) (450 pmols)for 3 hrs at 10× excess prior to tryptic digestion. Iodoacetamide wasused as the alkylating agent after compound incubation. A control sample(45 pmols) was also prepared which did not have the addition of (I-7).For tryptic digests a 5 ul aliquot (7.5 pmols) was diluted with 15 ul of0.1% TFA prior to micro C18 Zip Tipping directly onto the MALDI targetusing alpha cyano-4-hydroxy cinnamic acid as the matrix (5 mg/ml in 0.1%TFA:Acetonitrile 50:50).

As depicted in FIG. 8, the expected peptide (GCLLNFLR) to be modifiedwas immediately evident at MH+ of 1280.73. This is the mass to beexpected when compound I-7, with an adduct mass of 345.16, is added tothe peptide mass of 935.51. The peptide was also quite evident in thecontrol sample as modified by lodacetamide at MH+ of 992.56.Interestingly the iodoacetamide modified peptide was not evident in thedigest reacted with compound I-7 indicating that the reaction wascomplete. There was no evidence of any other modified peptide at MH+ of1985.93 (TIDEL VECEETFGR).

Evidence of compound I-7 was observed at MH+ of 346.32 in the low massrange of the spectra. The fragmentation spectra of the 346.32 peak didshow many diagnostic fragments that were apparent in the PSD spectra ofthe two modified peptides (See FIG. 8).

To further verify the presence of the modified peptides with compoundI-7, the peptides at MH+ of 1280.73 and 1985.93 were subjected to PSD(MS/MS) analysis. A correlation analysis with the homosapien databaseidentified the correct peptide modified by I-7.

Instrumental:

For tryptic digests the instrument was set in Reflectron mode with apulsed extraction setting of 2200. Calibration was done using the LaserBiolabs Pep Mix standard (1046.54, 1296.69, 1672.92, 2093.09, 2465.20).For CID/PSD analysis the peptide was selected using cursors to set iongate timing and fragmentation occurred at a laser power about 20% higherand He was used as the collision gas for CID. Calibration for fragmentswas done using the P14R fragmentation calibration for the Curved fieldReflectron.

Example 279

Omnia Assay Protocol for Potency Assessment Against Active Forms of ITKKinase

This example describes continuous-read kinase assays to measure inherentpotency of compound against active forms of ITK enzymes as described inExample 251 above except that the modified ITK-optimized reagentconditions are:

-   -   [ITK]=10 nM, [ATP]=25 μM, [Y6-Sox]=10 μM (ATP K_(Mapp)=33 μM).

Example 280

Table 14 shows the activity of selected compounds of this invention inthe ITK inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≤10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≥10,000 nM.

TABLE 14 ITK Inhibition Data Compound # ITK Inhibition I-1 C I-2 B I-4 BI-7 A I-27 B I-28 B I-33 A I-35 B I-38 B I-39 C I-40 A I-45 B I-54 BI-55 B I-56 C I-69 B I-70 A I-72 A I-73 A I-75 A I-76 A I-77 B I-78 AI-79 B I-80 A I-88 B I-89 B I-90 A I-91 B I-94 B I-95 B I-96 B I-97 BI-103 B I-105 B I-107 C I-108 B I-110 B I-114 D I-116 A I-118 B I-121 AI-122 A I-124 A I-125 B I-126 B I-128 A I-129 A I-131 A I-133 C I-134 BI-135 B I-138 B I-139 A I-140 B I-142 A I-143 B I-146 B I-147 A I-149 DI-150 A I-151 B I-152 A I-153 A I-154 A I-155 A I-156 A I-157 A I-158 BI-159 C I-160 A I-162 A I-163 D I-164 B I-165 A I-166 A I-167 A I-168 AI-169 A I-170 A I-172 B I-173 A I-174 A I-176 B I-177 B I-178 B I-180 CI-182 B I-183 C I-185 A I-186 A I-188 A I-189 A I-190 B I-192 A I-194 AI-195 B I-198 C I-199 B I-200 E I-201 C I-202 C I-204 B I-207 A I-208 BI-209 A I-210 B I-215 B I-217 B I-218 A I-219 B I-220 C I-227 A I-228 BI-230 A I-233 A I-237 A I-242 A I-243 B I-244 C I-245 A I-247 B I-248 AI-249 C I-313 B I-315 B I-316 B I-318 B I-321 C I-322 A I-324 A I-329 AI-333 B I-336 C I-337 B I-342 B I-353 B I-354 C I-355 B I-356 B I-357 BI-359 A I-362 B

Example 281

Omnia Assay Protocol for Potency Assessment Against Active Forms of BMXKinase

This example describes continuous-read kinase assays to measure inherentpotency of compound against active forms of BMX enzymes as described inExample 251 above except that the modified BMX-optimized reagentconditions are:

-   -   [BMX]=2.5 nM, [ATP]=100 μM, [Y5-Sox]=7.5 μM (ATP K_(Mapp)=107        μM).

Example 282

Table 15 shows the activity of selected compounds of this invention inthe BMX inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≤10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≥10,000 nM.

TABLE 15 BMX Inhibition Data Compound # BMX Inhibition I-4 A I-7 A I-27A I-28 A I-33 A I-35 A I-38 A I-39 A I-40 A I-45 A I-126 A I-128 A I-129A I-131 A I-133 A I-134 A I-135 A I-244 A I-245 A I-247 A I-248 A

Example 283

Cloning, Expression and Purification of EGFR-WT and EGFR C797S MutantUsing Baculovirus and Insect Cells

(i) Subcloning of EGFR-WT and Mutant Kinase Domains

Amino acids 696 to 1022 of the EGFR-WT kinase domain (NM_005228,NP_005219.2) was subcloned into the NcoI and HindIII sites of thepFastHTa vector (Invitrogen, Carlsbad, Calif.). To make the EGFR-mutantprotein, the cysteine at position 797 was changed to a serine using theStratagene QuikChange kit (Stratagene, Cedar Creek, Tex.), according tomanufacturer's instructions.

(ii) Expression

P1 baculovirus stocks were generated in SF9 cells via Blue Sky Biotech'ssuspension transfection protocol (Worcester, Mass.). Expression analysiswas conducted in 125 ml culture of SF21 insect cells ((grown in SF900ISFM (Invitrogen cat #10902-088), supplemented with 10 mg/L gentamicin(Invitrogen, Carlsbad, Calif., cat#15710-064)) using a viral load of 0.1ml of virus per 100 ml of cell suspension. Expression was optimizedusing Blue Sky Biotech's Infection Kinetics Monitoring system(Worcester, Mass.).

(iii) Purification

Infected insect cells were pelleted. Cell pellets were resuspended inBlue Sky Biotech's lysis buffer (Worcester, Mass., 1×WX; solubilizationbuffer, containing a protease inhibitor cocktail of leupeptin,pepstatin, PMSF, aprotinin and EDTA) at a ratio of 10 ml per gram of wetcell paste. Cells were lysed by sonication and the lysate was clarifiedby centrifugation at 9,000 RPM for 30 minutes in a GSA rotor. 500 μl bedvolume of NiNTA resin (Qiagen, Valencia, Calif.) was added to thesupernatants and batch bound for two hours with constant agitation. Thematerial was transferred by gravity into an empty 2 ml column. Thecolumn was washed with 2 ml of wash buffer (Blue Sky Biotech, Worcester,Mass., 1×WX, 25 mM imidazole). The protein was eluted with1×WX+imidazole at varying concentrations: Elution 1:75 mM imidazole (2fractions, 1 column volume); Elution 2:150 mM imidazole (2 fractions, 1column volume); Elution 3:300 mM imidazole (2 fractions, 1 columnvolume). All the elution fractions were analyzed by SDS page followed byCoomassie staining and Western Blotting using anti-penta-his antibody(Qiagen, Valencia, Calif.). The carboxy-terminal six-histidine “tag” wasremoved from some of the purified protein using AcTEV Protease kit(Invitrogen, Carlsbad, Calif., Cat#12575-015), following manufacturer'sinstructions. All the samples (pre- and post-Tev cut) were analyzed bySDS page followed by Coomassie staining and Western Blotting, asdescribed above.

Example 284

Mass Spectrometry for EGFR

EGFR wild type and EGFR (mutant C797S) is incubated with 10-fold excessof test compound for 1 hr and 3 hrs. 1 μl aliquots of the samples (totalvolume 5-8 ul) are diluted with 10 ul of 0.1% TFA prior to micro C4ZipTipping directly onto the MALDI target using Sinapinic acid as thedesorption matrix (10 mg/ml in 0.1% TFA:Acetonitrile 50:50). Intact massmeasurement reveals that the wild type has a nominal mass of about 37557and the mutant slightly lower at 37500. Reactivity is only observed forthe wild type EGFR with a new peak appearing at a mass consistent with asingle site covalent modification with test compound which has a mass of410 Da.

Example 285

Omnia Assay Protocol for Potency Assessment Against EGFR (WT) and EGFR(T790M/L858R) Active Enzymes

The Omnia Assay Protocol for potency assessment against EGFR isperformed as described in Example 251 above except that the EGFR-WT- andEGFR T790M/L858R-modified optimized reagent conditions are:

-   -   [EGFR-WT]=5 nM, [ATP]=15 mM, [Y12-Sox]=5 mM (ATP KMapp˜12 mM);        and [EGFR-T790M/L858R]=3 nM, [ATP]=50 mM, [Y12-Sox]=5 mM (ATP        KMapp˜45 mM).

Example 286

Tables 16 and 17 show the activity of selected compounds of thisinvention in the EGFR inhibition assay. Table 16 shows wild-type EGFRdata; Table 17 shows data for two EGFR mutants. The compound numberscorrespond to the compound numbers in Table 5. Compounds having anactivity designated as “A” provided an IC₅₀≤10 nM; compounds having anactivity designated as “B” provided an IC₅₀ 10-100 nM; compounds havingan activity designated as “C” provided an IC₅₀ of 100-1000 nM; compoundshaving an activity designated as “D” provided an IC₅₀ of 1000-10,000 nM;and compounds having an activity designated as “E” provided anIC₅₀≥10,000 nM.

TABLE 16 EGFR Wild Type Inhibition Data Compound # EGFR I-1 A I-2 B I-3A I-4 A I-5 A I-7 A I-8 A I-9 B I-10 B I-11 A I-23 C I-27 B I-28 B I-33A I-34 C I-35 B I-38 B I-39 D I-45 C I-56 B I^(R)-7 D I-54 B I-55 C I-60D I-69 B I-70 B I-71 B I-72 A I-74 C I-75 A I-76 A I-77 B I-78 A I-79 CI-80 B I-81 A I-82 A I-83 B I-84 A I-85 D I-86 D I-87 B I-88 B I-89 BI-90 A I-91 B I-92 B I-93 B I-94 C I-95 C I-96 B I-97 B I-98 C I-99 AI-100 D I-101 B I-102 C I-103 B I-104 A I-105 B I-106 D I-107 C I-108 CI-109 A I-110 B I-111 C I-112 B I-113 C I-114 D I-115 D I-116 A I-117 DI-118 B I-119 C I-120 A I-121 B I-122 B I-123 B I-124 B I-125 C I-126 BI-127 C I-128 B I-129 B I-130 B I-131 A I-132 B I-133 C I-134 B I-135 BI-136 C I-137 E I-138 B I-139 B I-140 B I-141 B I-142 B I-143 B I-144 AI-145 A I-146 D I-147 A I-148 D I-149 D I-150 A I-151 B I-152 B I-153 BI-154 B I-155 A I-156 A I-157 A I-158 B I-159 A I-160 A I-161 B I-162 AI-163 D I-164 B I-165 C I-166 C I-167 A I-168 B I-169 A I-170 B I-171 BI-172 C I-173 A I-174 A I-175 D I-176 A I-177 A I-178 A I-179 E I-180 CI-181 A I-182 A I-183 D I-184 A I-185 B I-186 A I-187 C I-188 A I-189 BI-190 C I-191 D I-192 B I-193 D I-194 B I-195 B I-196 D I-197 C I-198 AI-199 B I-200 A I-201 B I-202 C I-203 C I-204 B I-205 B I-206 D I-207 CI-208 B I-209 B I-210 B I-211 D I-212 D I-213 C I-214 C I-215 B I-216 DI-217 B I-218 A I-219 B I-220 C I-221 B I-222 C I-223 D I-224 C I-225 DI-226 D I-227 C I-228 B I-229 D I-230 B I-231 E I-232 D I-233 A I-234 EI-235 E I-236 D I-237 B I-238 E I-241 D I-242 B I-243 B I-244 C I-245 AI-246 D I-247 B I-248 A I-249 C I-277 B I-312 B I-313 B I-315 B I-316 BI-318 B I-321 C I-322 B I-323 E I-324 B I-325 D I-326 C I-327 B I-328 AI-329 B I-330 E I-331 D I-332 B I-333 B I-334 A I-335 A I-336 C I-337 BI-338 B I-339 A I-341 C I-342 C I-343 C I-344 C I-345 C I-346 C I-347 BI-348 B I-349 B I-350 A I-351 C I-352 A I-353 B I-354 C I-355 B I-356 CI-357 C I-358 C I-359 B I-360 A I-362 B I-363 A I-369 C I-370 A I-371 BI-372 B I-373 B I-374 A I-375 D I-376 B I-377 B I-378 B I-379 C I-381 AI-382 C

TABLE 17 EGFR mutant (T790M/L858R and T790M) Inhibition Data Compound #EGFR (T790M/L858R) EGFR (T790M) I-1 A — I-2 A A I-3 A A I-4 A A I-5 A —I-7 A — I-8 A — I-9 B — I-10 B — I-11 A — I-23 B — I-35 A — I-38 A —I-39 C — I-56 A B I^(R)-7 D — I-96 A A

Example 287

Cellular Assays for EGFR Activity

Compounds were assayed in A431 human epidermoid carcinoma cells using amethod substantially similar to that described in Fry, et al., Proc.Natl. Acad. Sci. USA Vol 95, pp 12022-12027, 1998. Specifically, A431human epidermoid carcinoma cells were grown in 6-well plates to 90%confluence and then incubated in serum-free media for 18 hr. Duplicatesets of cells were treated with 1 μM designated compound for 2, 5, 10,30, or 60 min. Cells were washed free of the compound with warmedserum-free medium, incubated for 2 hr, washed again, incubated another 2hr, washed again, and then incubated another 2 hr washed again andincubated for additional 2 hr and then stimulated with 100 ng/ml EGF for5 min. Extracts were made as described Fry, et al.

Compounds were assayed in A431 human epidermoid carcinoma cells using amethod substantially similar to that described in Fry, et al.Specifically, A431 human epidermoid carcinoma cells were grown in 6-wellplates to 90% confluence and then incubated in serum-free media for 18hr. Cells were then treated with 10, 1, 0.1, 0.01, or 0.001 μM testcompound for 1 hr. Cells were then stimulated with 100 ng/ml EGF for 5min, and extracts were made as described in Fry, et al. 20 ug totalprotein from lysates were loaded on gel and blots were probed for eitherEGFR phosphorylation or p42/p44 Erk phosphorylation.

Example 288

Washout Experiment for EGFR Activity

A431 human epidermoid carcinoma cells were grown in 6-well plates to'90% confluence and then incubated in serum-free media for 18 hr.Duplicate sets of cells were treated with 1 μM designated compound for 1hr. One set of cells was then stimulated with 100 ng/ml EGF for 5 min,and extracts were made as described. The other set of cells was washedfree of compound I-7 with warmed compound-free medium, incubated for 2hr, washed again, incubated another 2 hr, washed again, and thenincubated another 2 hr washed again and incubated for additional 2 hrand then stimulated with EGF. The results of this experiment withcompound I-7 are depicted in FIG. 10.

Example 289

Washout Experiment in HCC827 Cells Containing EGFR Deletion mutantHCC827

Cells (ATCC, Manassas, Va.) were plated in Growth Media (RPMI 1640)supplemented with 10% FBS, 10 uM HEPES, 2 mM 1-glutamine, 1 mMNaPyruvate and pen/strep (Invitrogen, Carlsbad, Calif.) at a density of2.5×10⁵ cells per well in 6 well tissue culture plates. Twenty fourhours later the cells were washed 2× with PBS then serum starvedovernight in Basal Media (Growth Media without FBS).

The following morning the media was removed and 2 ml fresh Basal Mediacontaining 1 uM compound in 0.1% DMSO was added to duplicate wells. At 1hour, one well of cells was treated with 100 ng/ml of EGF for 5 minutes,rinsed with PBS, then lysed by scraping into 75 ul of Cell ExtractionBuffer (Invitrogen, Carlsbad, Calif.) plus PhosSTOP PhosphataseInhibitor and Complete Protease Inhibitor (Roche, Indianapolis, Ind.)for the 0 h time point. The compound was removed from the second set ofwells and they were washed 2× with Basal Media. The cells were washedwith Basal Media every 2 hours until 8 hours when they were treated withEGF and lysed as at the 0 h time point.

Lysate protein concentrations were determined by BCA Assay (Pierce,Rockford, Ill.) and 10 ug of each lysate was separated by 4-12% gradientSDS-PAGE (Invitrogen), transferred to Immobilon-FL membrane (Millipore)and probed with rabbit anti-Phospho-EGFR (Tyr1068) (Zymed-nowInvitrogen) and mouse anti-EGFR (Cell Signaling Technologies, Danvers,Mass.) antibodies. Phospho-protein signals were quantitated usingOdyssey Infrared Imagning (Li-Cor Biosciences, Lincoln, Nebr.). Theresults of this experiment are depicted in FIG. 9 where it showscompound I-2 compared to results of compound I-4 and compound I-7 in thesame “washout” experiment.

Example 290

Mass Spectrometry for ERBB4

Erbb4 kinase domain (Upstate) was incubated with compound for 60 minutesat 10-fold excess of compound I-4 and I-11 to protein. 1 μl aliquots ofthe samples (total volume of 4.24 ul) were diluted with 10 μl of 0.1%TFA prior to micro C4 ZipTipping directly onto the MALDI target usingSinapinic acid as the desorption matrix (10 mg/ml in 0.1%TFA:Acetonitrile 50:50). For intact protein mass measurement theinstrument was set in Linear mode using a pulsed extraction setting of16,952 for the myoglobin standard used to calibrate the instrument(Shimadzu Axima TOF²).

Intact ErbB4 protein occurs at MH+ of 35850 with corresponding sinapinic(matrix) adducts occurring about 200 Da higher. A stochiometricincorporation of the test compound (I-4 and I-11) (Mw of 410 Da)produced a new mass peak which is approximately 410 Da higher (MH+ of36260). This is consistent with covalent modification of ErbB4 withcompounds I-4 and I-11.

Example 291

ErbB1, ErbB2 and/or ErbB4 Kinase Inhibition

Compounds of the present invention were assayed as inhibitors of one ormore of ErbB1, ErbB2, and/or ErbB4 in a manner substantially similar tothe method described by Invitrogen Corp (Invitrogen Corporation, 1600Faraday Avenue, Carlsbad, Calif., CA;http://www.invitrogen.com/downloads/Z-LYTE_Brochure_1205.pdf) using theZ'-LYTE™ biochemical assay procedure or similar biochemical assay. TheZ'-LYTE™ biochemical assay employs a fluorescence-based, coupled-enzymeformat and is based on the differential sensitivity of phosphorylatedand non-phosphorylated peptides to proteolytic cleavage. Using thisassay, Compound I-56 was found to inhibit ERBB1 with an IC₅₀ of 2,233nM. Using this assay, Compound I-56 was found to inhibit ERBB4 (HER4)with an IC₅₀ of 2,165 nM.

Example 292

Mass Spectrometry for Janus-3 Kinase (JAK3)

JAK3 kinase (33 pmols; Invitrogen) was incubated with (I-7) (327 pmols)for 3 hrs at 10× excess prior to tryptic digestion. Iodoacetamide wasused as the alkylating agent after compound incubation. For trypticdigests a 5 ul aliquot (5.5 pmols) was diluted with 15 ul of 0.1% TFAprior to micro C18 Zip Tipping directly onto the MALDI target usingalpha cyano-4-hydroxy cinnamic acid as the matrix (5 mg/ml in 0.1%TFA:Acetonitrile 50:50).

As depicted in FIG. 11, the expected peptide (LVMEYLPSGCLR) to bemodified was immediately evident as the largest peak at MH+ of 1725.88.This is the mass to be expected when compound I-7, with an adduct massof 345.16, is added to the peptide mass of 1380.70. Interestingly theiodoacetamide modified peptide was not evident at MH+ of 1437.73 in thedigest reacted with compound I-7 indicating that the reaction was notentirely complete. There was also evidence for a number of othermodified peptides, however, their signals were low.

Evidence of compound I-7 was observed at MH+ of 346.12 in the low massrange of the spectra. The fragmentation spectra of the 346.12 peak didnot show diagnostic fragments that were apparent in the PSD spectra ofthe modified peptides (See FIG. 11).

To further verify the presence of the modified peptides with compoundI-7, the peptides at MH+ of 1725.88 and 1118.55 were subjected to PSD(MS/MS) analysis. A correlational analysis with the homosapien databaseidentified the correct peptides as being modified by I-7. Compound I-11was also tested using the same procedure and showed measurablemodification.

Instrumental:

For tryptic digests the instrument was set in Reflectron mode with apulsed extraction setting of 2200. Calibration was done using the LaserBiolabs Pep Mix standard (1046.54, 1296.69, 1672.92, 2093.09, 2465.20).For CID/PSD analysis the peptide was selected using cursors to set iongate timing and fragmentation occurred at a laser power about 20% higherand He was used as the collision gas for CID. Calibration for fragmentswas done using the P14R fragmentation calibration for the Curved fieldReflectron.

Example 293

Omnia Assay Protocol for Potency Assessment Against the Active Form ofJAK3:

The Omnia Assay Protocol for potency assessment against JAK3 wasperformed in a substantially similar manner as that described in Example251 above except that the modified JAK3-optimized reagent conditionswere:

-   -   [JAK3]=5 nM, [ATP]=5 μM, [Y12-Sox]=5 μM (ATP KMapp˜5 μM).

Example 294

Table 18 shows the activity of selected compounds of this invention inthe JAK3 inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≤10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≥10,000 nM.

TABLE 18 JAK3 Inhibition Data Compound # JAK3 Inhibition I-1 A I-2 A I-3A I-4 A I-5 A I-7 A I-8 A I-9 A I-10 B I-11 A I-23 A I-27 B I-28 A I-33A I-34 B I-35 A I-38 B I-39 A I-40 A I-45 A I-56 B I^(R)-7 D I-96 AI-182 A I-238 D I-241 C I-242 A I-243 A I-244 A I-245 A I-246 C I-247 AI-248 A I-323 E I-360 A

Example 295

JAK3 Cellular Assay Protocol in CTLL2 Cells

Compounds I-2, I-4 and I-7 were tested in the following protocol. CTLL2:murine lymphoma cell line ATCC: TIB-214. 5×10⁶ cells/sample were IL-2starved in RPMI-1640 media for 2 hours. Designated samples were thentreated with compound for 90 minutes. Samples, except DMSO control werethen stimulated with 100 nM IL-2 for 10 minutes. Samples were lysed andsubjected to Western Analysis. The results are displayed in FIG. 12,FIG. 13 and FIG. 14.

Example 296

BTK Occupancy in Ramos Cells with I-7 and I-215 Using Streptavidin Beads

Ramos cells were incubated with 0.1, 0.05, 0.01, or 0.001 μM I-7 inserum free media for 1 hour at 37° C. The cells were pelleted bycentrifugation and lysed in Cell Extraction buffer (Invitrogen) for 10minutes on ice, centrifuged (10 minutes at 14,000 rpm) and thesupernatant was collected. Cell lysates were incubated with 1 μM I-215for 1 hour at room temperature, then incubated with streptavidin-coupledagarose beads (ThermoFisher) overnight at 4° C. The beads were washedthree times with lysis buffer and the bound proteins were boiled off thebeads at 95° C. for 5 minutes in 4×LDS Sample Buffer. The amount of BTKassociated with the probe I-215 was assessed by BTK western blot. Allvalues were normalized to the DMSO-treated sample which is set to 100%.FIG. 16 shows the western blot; FIG. 17 shows quantitation of FIG. 16demonstrating unoccupied BTK protein is available to the probe I-215when the cells have been exposed to low concentrations (10 nM, 1 nM) ofI-7 but at higher concentrations of I-7 the BTK protein is fullyoccupied and cannot interact with 1-215.

Example 297

Washout Experiment with I-7 and Probe Compound I-215

Ramos cells were incubated with 0.1 μM I-7 or a reversible BTK inhibitorcontrol compound in serum free media for 1 hour at 37° C. The cells werethen washed in compound-free media and lysed 0, 4, 6, or 8 hours aftercompound removal. Cell lysates were incubated with 1 μM I-215 for 1 hourat room temperature, then overnight at 4° C. with streptavidin-coupledagarose beads. Protein was boiled off the beads and BTK association wasassessed by western blot. FIG. 18 shows the Western blot; FIG. 19 showsthe quantitation of FIG. 18 and demonstrates all the BTK protein remainsoccupied by I-7 for over 8 hours. This suggests that the timeframe forre-synthesis of detectable BTK protein in Ramos cells is greater than 8hours. In contrast, with the reversible inhibitor control, 45% of BTKprotein is unbound and available to the probe at 0 hours and by 4 hours100% of BTK protein is unbound and available to bind the probe. Allsamples were normalized to the DMSO-treated cells harvested at 0 hours.

Example 298

Measuring BTK Occupancy from In Vitro Samples by ELISA

In order to determine the amount of free BTK in cell or tissue lysates,an ELISA protocol was employed that utilizes a biotinylated probecompound that binds only to free, unoccupied BTK. The conjugated biotinis captured on a streptavidin-coated ELISA plate and detected with amouse anti-BTK antibody (Becton Dickinson, Franklin Lakes, N.J., USA)and a secondary goat anti-mouse HRP antibody (Zymed, South SanFrancisco, Calif., USA).

All samples were prepared with equal concentrations of Biorad lysisbuffer (Hercules, Calif., USA), 0.5% bovine serum albumin in PBS with0.05% Tween-20 to give a final concentration of 1 μM I-215. Samples wereincubated in a mixing plate for 1 hr at room temperature while shakingto allow probe compound I-215 to bind to free BTK. After incubation with1-215, samples were added to a washed, streptavidin-coated ELISA plate(Pierce, Rockford, Ill., USA) and incubated for 1 hr at room temperaturewhile shaking. The plate was then washed with PBS containing 0.05%Tween-20 using an automatic plate washer. Anti-BTK antibody was preparedat 1:1000 dilution in 0.5% BSA in PBS (0.05% Tween-20) and added to theELISA plate. The plate was incubated for 1 hr at room temperature whileshaking. The plate was washed as described above and the secondary HRPantibody was prepared at 1:5000 dilution in 0.5% BSA in PBS (0.05%Tween-20). The plate was incubated and washed as described above. TMBwas added to the plate, and OD₆₅₀ was monitored until reaching 1 ODunit. The reaction was then stopped with addition of H₂SO₄. The platewas analyzed using Gen 5 software, and a 4 Parameter Logistic curve wasemployed to quantitate samples. Recombinant BTK (Invitrogen, Carlsbad,Calif., USA) was used for the standard curve.

Table 19 shows results with Ramos cells reported as concentration atwhich >50% or >90% of BTK is occupied. A concentration designated as “A”is greater than 1 nM; a concentration designated as “B” is greater than10 nM; and a concentration designated as “C” is greater than 50 nM.

TABLE 19 Compound # >50% occupancy >90% occupancy I-7 A B I-182 A C I-96A B

Example 299

Human Primary B Cell Covalent Probe Occupancy In Vitro

Human primary B cells were isolated as described in Example 256, thenresuspended in RPMI media (10% serum). The compound to be analyzed wasadded at a 1:1000 dilution to media. Cells were incubated with compoundin a tissue culture incubator for 1 h at 37° C. After incubation, thecells were pelleted, washed with 1×PBS, and lysed on ice for 45 min withoccasional agitation. Samples were spun in a chilled microcentrifuge for30 min at 14,000 rpm and the supernatant was isolated. The supernatantwas analyzed as described in Example 283 using I-215. I-96 and I-182occupied at least 50% of BTK at concentrations greater than 10 nM.

Example 300

Dog Primary B Cell Covalent Probe Occupancy In Vitro

Canine whole blood (30 mL) was diluted to 50 mL total with 1×PBS andlayered on top of Histopaque-1077 (Sigma Aldrich). The wholeblood-Histopaque was spun at 400×g for 30 min in a Beckman centrifugewith no brake. Peripheral blood mononuclear cells (PBMCs) were collectedand pelleted at 400×g for 15 min. Red blood cells (RBCs) were lysed with2.5 mL RBC lysis buffer (Boston Bioproducts) and the remaining PBMCswere washed 3 times in 1×PBS at 250×g. PBMCs were treated with compoundat a 1:1000 dilution for one hour at 37° C., washed with PBS and lysedon ice for 45 minutes. The lysate was centrifuged for 30 minutes at14,000×g and the supernatant collected. The supernatant was analyzed asdescribed in Example 283 using I-215. I-96 occupied at least 50% of BTKat concentrations greater than 10 nM.

Example 301

Measuring BTK Occupancy from In Vivo Samples by ELISA

Rats were dosed orally with 30 mg/kg of compound and spleens wereharvested either 2 or 24 hours after compound treatment. Rat spleenswere disrupted between two microscope slides coated with frosted glassto recover single cell suspensions. Red blood cells were lysed byincubating with RBC lysis buffer (Boston BioProducts) for 2 minutes atroom temperature, the cells were then resuspended in RPMI complete mediaand pelleted by centrifugation. Rat B cells were isolated by positiveselection with B220+ antibody-magnetic bead conjugates, purified by MACScolumn and lysed in Bio-Rad lysis buffer at a concentration of 10million cells/100 μl. Lysates were analyzed employing the biotinylatedprobe compound I-215 in an ELISA protocol as described in detail inExample 278. Table 20 shows the results.

TABLE 20 Treatment % BTK Occupancy 2 h % BTK Occupancy 24 h Vehicle 0 0I-96 87 60 I-4 87 68 I-7 98 78 I-190 18 13 I-182 99 79

Example 302

Proteomics Analysis

Proteins that are covalently bound to I-215 in a cell lysate areidentified using mass spectrometry. Cell lysate is incubated with 1 μMI-215 for 1 hour at room temperature, followed by the addition ofstreptavidin-coupled agarose beads. Mass spectrometry is used toidentify proteins other than BTK. These are potential “off-target”interactions.

While a number of embodiments of this invention are described herein, itis apparent that the basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

We claim:
 1. A method for inhibiting the activity of one or moreTEC-kinases, or a mutant thereof, in a patient or in a biological samplecomprising the step of administering to said patient or contacting saidbiological sample with a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 2. The method accordingto claim 1, wherein the activity of the TEC-kinase, or a mutant thereof,is inhibited irreversibly.
 3. The method according to claim 2, whereinthe TEC-kinase is selected from one or more of TEC, ITK or BMX.
 4. Themethod according to claim 3, where the activity of the one or more ofTEC, ITK or BMX is inhibited irreversibly by covalently modifying Cys449 of TEC, Cys 442 of ITK or Cys 496 of BMX.